Nucleic acids for detection and discrimination of genotypes of Chlamydophila psittaci

ABSTRACT

Methods of detecting  Chlamydophila , including differentiating between species of  Chlamydophila  and/or strains of  Chlamydophila psittaci  are disclosed, for example to detect and genotype a  Chlamydophila psittaci  infection. A sample suspected of containing a nucleic acid of a  Chlamydophila , is screened for the presence of that nucleic acid. The presence of the  Chlamydophila  nucleic acid indicates the presence of the  Chlamydophila  bacterium. Determining whether a  Chlamydophila  nucleic acid is present in a sample can be accomplished by detecting hybridization between a  Chlamydophila  specific primer, a  Chlamydophila psittaci  specific primer, and/or a  Chlamydophila psittaci  genotype-specific primer and the  Chlamydophila  nucleic acid containing sample. Thus, primers for the detection, species-specific and/or genotype-specific identification of  Chlamydophila psittaci  are disclosed. Kits that contain the disclosed primers also are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US2010/036742, filed May 28, 2010, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/182,628, filed May 29, 2009, which isincorporated by reference herein in its entirety.

FIELD

This disclosure relates to nucleic acids for the detection andidentification of Chlamydophila psittaci, as well as kits includingprimers and methods of using the primers to detect, diagnose andgenotype Chlamydophila psittaci.

BACKGROUND

Chlamydophila psittaci (C. psittaci) is an intracellular pathogen and amember of the Chlamydiaceae family that is most frequently associatedwith Psittaciformes. C. psittaci can infect 465 avian species in 30avian orders, but the at least 153 species in the order Psittaciformescan also infect a wide range of mammalian hosts. C. psittaci has theability to remain infectious in the environment for months, which cancause economically devastating outbreaks in poultry farms andrespiratory disease (psittacosis) in both mammals and birds.Transmission of this respiratory pathogen can occur through directcontact with infected birds, bird feces, nasal discharges, and aerosols.Zoonotic infections in humans usually result from close contact withinfected captive birds, companion, or free-ranging birds; human-to-humantransmission has also been suggested. Regardless of the transmissionmethod, infection may lead to severe pneumonia and a wide spectrum ofother medical complications. From 1988 through 2003, a total of 935human cases of psittacosis were reported to the U.S. Centers for DiseaseControl and Prevention; most were related to contact withPsittaciformes. Approximately 100 psittacosis cases are reportedannually in the United States, and one person may die of this diseaseeach year. Individuals with occupations associated with commercialpoultry as well as those with routine contact with companion or aviarybirds are considered most at risk for infection. Laboratory-acquiredinfections also remain a concern.

C. psittaci is currently grouped into seven avian genotypes (A through Fand the recently identified genotype, E/B) and two non-avian genotypes(M56 and WC). Recent reclassification of C. psittaci has resulted in theseparation of C. abortus and C. caviae into distinct species, althoughthese species are genetically closely related.

There is a need for methods to quickly and reliably detect C. psittaci.This will aid in treatment, as quick diagnosis will improve treatmentoutcomes.

SUMMARY

Disclosed herein are methods for the detection and identification of C.psittaci. In several embodiments, the methods can detect and identifyeach of the individual avian C. psittaci genotypes. In addition, themethods can be used to identify and discriminate between closely relatedstrains, such as C. caviae and C. abortus. The methods are useful fordetermining the existence of genetic variants within the C. psittacispecies. The versatility of the methods also makes it useful in manyapplications, such as, but not limited to, (i) improving the timelyreporting of results to facilitate epidemiological investigations; (ii)pathogenicity and transmission studies; (iii) prospective screening ofcompanion birds or livestock; (iv) current and retrospective analysis ofspecimens collected during an outbreak of C. psittaci; and (v) a greatercharacterization of this pathogen, leading to a better understanding ofC. psittaci infection within human and avian populations. In someembodiments, the disclosed methods contribute to the development of anall-inclusive molecular typing system for this pathogen. In additionalembodiments, these methods will also provide valuable information fordesigning public health measures during a C. psittaci outbreak.

In particular examples, the methods for the detection and identificationof Chlamydophila involve direct dection of a hybridized primer or probe,such as by Southern blot or dot blot analysis. In other examples,hybridized primers or probes are further used to direct amplification ofa target Chlamydophila nucleic acid, which is then detected using alabel such as a self-quenching fluororophore or by hybridization of alabeled probe to the amplified product.

In some embodiments, primers are provided that are specific for theamplification of a Chlamydophila nucleic acid. In other embodiments, thepresent disclosure relates to primers that are specific for theamplification of a C. psittaci nucleic acid. These primers are 15 to 40nucleotides in length and include a nucleic acid set forth as SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:8, and are capable of directing amplification of a Chlamydophilapsittaci nucleic acid in a sample. In another embodiment, the primersinclude 15 to 40 nucleotides of a nucleic acid sequence at least 95%identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7 or SEQ ID NO: 8. In yet another example, the primers include,or consist of a nucleic acid sequence that consists essentially of, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQID NO: 8.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a generalized procedure forhybridizing and amplifying C. psittaci nucleic acids with C. psittacispecific LUX™ primers.

FIG. 2 is a phylogenetic tree of the ompA gene.

FIGS. 3A-3C are High-resolution Melt (HRM) analysis curves obtained fromthe melting of a sample containing Chlamydophila nucleic acid amplifiedwith Chlamydophila specific primers. FIG. 3A is a HRM analysis curveobtained from the melting of a sample containing Chlamydophila nucleicacids with a Chlamydophila-specific primer. FIG. 3B is a HRM analysiscurve obtained from the melting of a sample containing C. psittacinucleic acids with a C. psittaci-specific primer. FIG. 3C is a HRM curveobtained from the melting of a sample containing C. psittaci nucleicacids with a C. psittaci genotype F-specific primer.

FIGS. 4A-4B is a sequence alignment of C. psittaci genotypes. FIG. 4A isa sequence alignment of Ppac amplicons and shows the consensus sequence(SEQ ID NO: 18). Only the sequence of the genotype C Ppac amplicon (SEQID NO: 19) was divergent from the consensus sequence. FIG. 4B is asequence alignment of GTpc amplicons and shows the consensus sequence(SEQ ID NO: 20). The genotype A-F GTpc amplicon sequences (SEQ ID NOs21-26) are also shown and are highly divergent from the consensussequence. A dash (-) indicates identical sequences; a * indicates noconsensus sequence. Each genotype (**) is presented by reference strainsand, where applicable, specimen sequences. Genotype A includes DD34 andspecimens 25 and 83; genotype B includes CP3 and specimens 30 and 31;genotype C includes CT1, genotype D includes NJ1, genotype E includesVr-122 and specimens 3 and 5, and genotype F includes VS-225.Underscored and bold portions of the sequences are primer bindinglocations.

FIGS. 5A-5B are graphs of Real-Time Polymerase Chain Reaction dataobtained from the amplification of samples containing Chlamydophilapsittaci nucleic acids amplified with Chlamydophila caviae specificprimers. FIG. 5A is an amplification plot for the amplification of C.psittaci nucleic acids using C. caviae specific primers. All of the C.psittaci samples were observed to amplify below the threshold valueconsidered sufficient for amplification of C. caviae nucleic acids. FIG.5B discloses an amplification plot for samples that were archived withthe University of Georgia as being C. caviae positive. The majority ofsamples were found to amplify above the threshold value sufficient to bepositively identified as a C. caviae nucleic acid. It is likely thatlong-term storage conditions and/or solutions used during thepreparation of some of the original samples adversely affected thosetest samples, resulting in amplification levels lower than the thresholdvalue and thus no determination of bacterial source could be made.

FIG. 6 is a flow chart showing an example of the detection ofChlamydophila species (FIG. 6A) and the differentiation of Chlamydophilapsittaci genotypes A-F (FIG. 6B).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and one letter code for amino acids, as defined in 37 C.F.R.§1.822. If only one strand of each nucleic acid sequence is shown, thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an 18.9 KB ASCIItext file named “seq listing_(—)82676-02.txt,” which was last modifiedon May 21, 2010, and which is incorporated by reference herein.

SEQ ID NO: 1 is the nucleotide sequence of a theoretical oligo.

SEQ ID NO: 2 is the nucleotide sequence of a theoretical oligo.

SEQ ID NO: 3 is the nucleotide sequence of a C. psittaci ompA (Ppac)forward real-time PCR primer.

SEQ ID NO: 4 is the nucleotide sequence of a C. psittaci ompA (Ppac)reverse real-time PCR primer.

SEQ ID NO: 5 is the nucleotide sequence of a C. psittaci ompA (GTpc)forward real-time PCR primer.

SEQ ID NO: 6 is the nucleotide sequence of a C. psittaci ompA (GTpc)reverse real-time PCR primer.

SEQ ID NO: 7 is the nucleotide sequence of a C. psittaci ompA (GT-F)forward real-time PCR primer.

SEQ ID NO: 8 is the nucleotide sequence of a C. psittaci ompA (GT-F)reverse real-time PCR primer.

SEQ ID NO: 9 is the nucleotide sequence of a C. psittaci ompA (ompA)forward real-time PCR primer.

SEQ ID NO: 10 is the nucleotide sequence of a C. psittaci ompA (ompA)reverse real-time PCR primer.

SEQ ID NO: 11 is an exemplary nucleotide sequence of the C. psittaciMOMP gene (GENBANK® Accession no. X56980).

SEQ ID NO: 12 is an exemplary nucleotide sequence of the C. psittaciompA gene (GENBANK® Accession no. AY762608).

SEQ ID NO: 13 is an exemplary nucleotide sequence of C. psittaci ompA(GENBANK® Accession no. AY762612).

SEQ ID NO: 14 is an exemplary nucleotide sequence of C. caviae ompA(GENBANK® Accession no. AF269282).

SEQ ID NO: 15 is an exemplary nucleotide sequence of C. abortus ompA(GENBANK® Accession no. CR848038).

SEQ ID NO: 16 is the nucleotide sequence of a C. caviae ompA reversereal-time PCR reverse primer.

SEQ ID NO: 17 is the nucleotide sequence of a C. caviae ompA forwardreal-time PCR forward primer.

SEQ ID NO: 18 is the consensus nucleotide sequence of the Ppac ampliconof C. psittaci.

SEQ ID NO: 19 is the nucleotide sequence of the Ppac amplicon of C.psittaci Genotype C.

SEQ ID NO: 20 is the consensus nucleotide sequence of the GTpc ampliconof C. psittaci.

SEQ ID NO: 21 is the nucleotide sequence of the GTpc amplicon of C.psittaci Genotype A.

SEQ ID NO: 22 is the nucleotide sequence of the GTpc amplicon of C.psittaci Genotype B.

SEQ ID NO: 23 is the nucleotide sequence of the GTpc amplicon of C.psittaci Genotype C.

SEQ ID NO: 24 is the nucleotide sequence of the GTpc amplicon of C.psittaci Genotype D.

SEQ ID NO: 25 is the nucleotide sequence of the GTpc amplicon of C.psittaci Genotype E.

SEQ ID NO: 26 is the nucleotide sequence of the GTpc amplicon of C.psittaci Genotype F.

DETAILED DESCRIPTION

Several standard Polymerase Chain Reaction (PCR) techniques have beendeveloped for the detection and identification of C. psittaci. Most ofthem target major outer membrane protein (MOMP) genes. Identificationand genotyping of C. psittaci in avian samples and isolates is currentlyachieved by serological testing and molecular methods, such as outermembrane protein A (ompA) gene sequencing, restriction fragment lengthpolymorphism (RFLP), and microarray analysis. Traditionally, sequenceanalysis of the ompA gene has been considered the most accurate methodfor identifying all known genotypes. For diagnosis in humans,serological testing is rarely performed because the procedure islabor-intensive and requires specialized laboratory expertise andequipment. Thus, accurate diagnosis of C. psittaci infection is oftendelayed or missed and may result in improper treatment for patients.Diagnosis by molecular techniques, such as real-time PCR, is not readilyavailable in most public health laboratories, forcing them to rely uponinsensitive complement fixation or micro-immunofluorescence tests fordetecting C. psittaci antibodies in suspect cases. Since both complementfixation and micro-immunofluorescence require acute andconvalescent-phase sera, they are retrospective assays that areconsidered inadequate for a timely diagnosis.

Other newly developed molecular methods have improved upon thetraditional approaches of ompA sequencing and RFLP to genotype C.psittaci. These methods include DNA microarrays, real-time PCR assaysfor detecting amplified product using minor-groove binding probes andcompetitor oligonucleotides, and a multilocus variable-number tandemrepeat analysis. However, there exists no rapid, simple and inexpensiveprocedure which can effectively discriminate among the known genotypesof C. psittaci and have the capability of identifying new strains inview of the significant genetic heterogeneity found within this species.Hence the need remains for a reliable and rapid assay for detecting andgenotyping C. psittaci, so that diagnosis is completed in sufficienttime to permit effective treatment of an infected subject.

The disclosed methods detect and identify C. psittaci. In particularexamples, the methods for the detection and identification ofChlamydophila involve direct dection of a hybridized primer or probe,such as by Southern blot or dot blot analysis. In other examples,hybridized primers or probes are further used to direct amplification ofa target Chlamydophila nucleic acid, which is then detected using alabel such as a self-quenching fluororophore. In several embodiments,the methods can detect and identify each of the individual avian C.psittaci genotypes. In addition, the methods can identify anddiscriminate between closely related strains, such as C. caviae and C.abortus, and determine the existence of genetic variants within the C.psittaci species. An example of the methods of distinguishing betweenChlamydophila species is presented in FIG. 6A.

In one aspect, the disclosure relates to primers that are specific forthe hybridization to and amplification of a Chlamydophila nucleic acid.Primers are disclosed that are specific for the amplification of a C.psittaci nucleic acid. In some embodiments, these primers include anucleic acid set forth as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, which are capable of directingamplification of a Chlamydophila psittaci nucleic acid in a sample. Inanother embodiment, the primers are at least 95% or 98% identical tothese disclosed sequences, or consist essentially of one of SEQ ID NOs:3-8

In some embodiments, the primers are capable of hybridizing to andamplifying a Chlamydophila nucleic acid, such as a C. psittaci nucleicacid or the nucleic acid of a particular C. psittaci genotype. Inseveral embodiments, the primers are between 15 and 40 nucleotides inlength and are capable of hybridizing under very high stringencyconditions to the complement of nucleic acids of C. psittaci genotypesA, B, C, D, E or F. In another aspect, the primers are capable ofhybridizing under very high stringency conditions to the complement ofnucleic acids of Chlamydophila psittaci genotypes A, B, C or E andinclude a nucleic acid sequence at least 95% identical to primers setforth as SEQ ID NO: 5 or SEQ ID NO: 6. In yet another aspect, thedisclosure relates to primers that are capable of hybridizing under veryhigh stringency conditions to a nucleic acid of Chlamydophila psittacigenotype F and include a nucleic acid sequence at least 95% identical toprimers set forth as SEQ ID NO: 7 or SEQ ID NO: 8.

In some aspects, the C. psittaci species-specific primers are a pair ofprimers, and the pair of primers is capable of hybridizing to anddirecting the specific amplification of complementary C. psittacinucleic acids. In one aspect, the pair of primers include one or moreforward primers with a nucleic acid sequence set forth as SEQ ID NO: 3,SEQ ID NO: 5, or SEQ ID NO: 7; and one or more reverse primers with anucleic acid sequence set forth as SEQ ID NO: 4, SEQ ID NO: 6, or SEQ IDNO: 8. In another aspect, the pair of primers include one or moreforward primers with a nucleic acid sequence at least 95% identical to anucleic acid sequence set forth as SEQ ID NO: 3, SEQ ID NO: 5, or SEQ IDNO: 7; and one or more reverse primers with a nucleic acid sequence atleast 95% identical to a nucleic acid sequence set forth as SEQ ID NO:4, SEQ ID NO: 6, or SEQ ID NO: 8. In yet another aspect, the pair ofprimers include one or more forward primers with a nucleic acid sequencethat consists essentially of or consists of SEQ ID NO: 3, SEQ ID NO: 5,or SEQ ID NO: 7; and one or more reverse primers with a nucleic acidsequence that consists essentially of SEQ ID NO: 4, SEQ ID NO: 6, or SEQID NO: 8. In some embodiments, the pair of primers specific for thedetection and identification of C. psittaci nucleic acids in a sampleare 15 to 40 nucleotides in length and include a nucleic acid sequenceat least 95% identical to the nucleotide sequence set forth as SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ IDNO: 8.

In another embodiment, the disclosure relates to primers capable ofhybridizing to and amplifying a Chlamydophila nucleic acid, such as a C.caviae or C. abortus nucleic acid. In some embodiments, the primers arebetween 15 and 40 nucleotides in length and are capable of hybridizingunder very high stringency conditions to a C. caviae or C. abortusnucleic acid in a sample. In one embodiment, the primers capable ofhybridizing to and amplifying a C. caviae nucleic acid include orconsist of a nucleic acid sequence set forth as SEQ ID NO: 16, or SEQ IDNO: 17. In another embodiment, the primers capable of hybridizing to andamplifying a C. caviae nucleic acid include a nucleic acid sequence atleast 95% identical to a nucleic acid sequence set forth as SEQ ID NO:16, or SEQ ID NO: 17, or consists essentially of SEQ ID NO: 16, or SEQID NO: 17.

In some embodiments, the nucleic acids are genotype-specific for thedetection and identification of C. psittaci genotypes, and are used inmethods of detecting and/or discriminating between C. psittacigenotypes. In one aspect, detecting hybridization of a nucleic acidsequence at least 95% identical to SEQ ID NO: 3 or SEQ ID NO: 4 with asample suspected of containing a Chlamydophila nucleic acid indicatesthe presence of C. psittaci, C. caviae or C. abortus in the sample. Inanother example, detecting hybridization of a nucleic acid sequence atleast 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6 with a samplesuspected of containing a Chlamydophila nucleic acid indicates thepresence of C. psittaci in the sample, such as C. psittaci genotype A,B, C, D, E or F, or a combination thereof. In yet another example,detecting hybridization of a nucleic acid sequence at least 95%identical to SEQ ID NO: 7 or SEQ ID NO: 8 with a sample suspected ofcontaining a Chlamydophila nucleic acid indicates the presence of C.psittaci genotype F. In one example, detecting hybridization of anucleic acid sequence at least 95% identical to SEQ ID NO: 16 or SEQ IDNO: 17 with a sample suspected of containing a Chlamydophila nucleicacid indicates the presence of C. caviae.

As demonstrated by the example presented in FIGS. 6A and B, methods arealso disclosed for detecting in a sample the presence of Chlamydophila,such as Chlamydophila psittaci (C. psittaci), for example in abiological sample obtained from a subject. The disclosed methods can beused for diagnosing a C. psittaci infection or confirming diagnosis of aC. psittaci infection in a subject by analyzing a biological specimenfrom the subject and detecting the presence of C. psittaci nucleic acidsand/or the specific C. psittaci genotype in the sample. Alternatively,the method can be used to quickly discriminate between Chlamydophilaspecies, such as C. psittaci, C. caviae and C. abortus.

In some embodiments, the detection method involves contacting abiological sample suspected of containing a C. psittaci nucleic acidwith a C. psittaci species-specific or genotype-specific primer orprobe, and detecting hybridization between the C. psittaci nucleic acidin the sample and the C. psittaci primer or probe. In some embodiments,the primer is detectably labeled for instance with a fluorophore. In oneembodiment, the method involves amplifying C. psittaci nucleic acidspresent in a sample, for example by Polymerase Chain Reaction (PCR)techniques. In another aspect, the method involves amplifying C.psittaci nucleic acids present in a sample with a C. psittaci specificprimer in combination with high-resolution melt (HRM) analysis tospecifically detect, identify and genotype C. psittaci nucleic acids inthe sample. In some embodiments, the primer is a C. psittacispecies-specific primer with a nucleic acid sequence set forth as SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ IDNO: 8, or a nucleic acid sequence that is 95% identical thereto.

In a further embodiment, the detection method relates to primers orprobes that are C. psittaci genotype specific. These methods includecontacting a sample suspected of containing a C. psittaci nucleic acidwith a primer or probe specific for a C. psittaci genotype and detectinghybridization between the C. psittaci nucleic acids in the sample andthe C. psittaci genotype-specific primer. Detection of the hybridizationbetween the C. psittaci genotype-specific primer or probe and the sampleindicates that C. psittaci nucleic acids and at least one C. psittacigenotype is present in the sample. In some embodiments, the primer orprobe is specific for the detection and identification of one or more C.psittaci genotypes. In another embodiment, the C. psittaci primer orprobe is specific for the detection and identification of a singlegenotype selected from C. psittaci genotypes A, B, C, D, E or F. Inother embodiments, the C. psittaci primer or probe is specific for theidentification of C. psittaci genotypes A, B, C and E. In a furtherembodiment, the C. psittaci primer or probe is specific for theidentification of C. psittaci genotype F.

The disclosure provides panels of primers that permit rapid evaluationof a subject with an apparent illness by quickly determining whether theillness is caused by C. psittaci. This rapid evaluation involves rulingout the presence of other bacterial, viral and Chlamydophila species(for example, by positively identifying a C. psittaci nucleic acid or C.psittaci genotype).

In one embodiment, the methods include amplifying the nucleic acids of asample with at least one primer specific for C. psittaci to diagnose aC. psittaci infection. In some embodiments, the primer specific for C.psittaci is 15 to 40 nucleotides in length and includes a self-quenchingdetectable label. In one example, a pair of primers specific for thedetection of C. psittaci or diagnosis of a C. psittaci infectionincludes a labeled primer and an unlabeled primer. In another example,nucleic acids amplified in a sample suspected of containingChlamydophila with a Chlamydophila specific primer are subjected tohigh-resolution melt analysis to discriminate between the Chlamydophilaspecies. In one example, high-resolution melt analysis of the amplifiednucleic acids can be used to genotype C. psittaci nucleic acids in thesample. In another example, high-resolution melt analysis of theamplified nucleic acids can be used to discriminate between C. psittaci,C. caviae, or C. abortus nucleic acids in a sample.

In some embodiments, the primers of the disclosure are capable ofhybridizing under very high stringency conditions to a C. psittaci, C.caviae or C. abortus nucleic acid. In one embodiment, the primer iscapable of hybridizing under very high stringency conditions to anucleic acid sequence set forth as SEQ ID NO:13, SEQ ID NO:14, or SEQ IDNO:15. In another embodiment, a C. caviae specific primer is capable ofhybridizing under very high stringency conditions to a nucleic acidsequence set forth as SEQ ID NO: 14. In yet another embodiment, a C.abortus specific primer is capable of hybridizing under very highstringency conditions to a nucleic acid sequence set forth as SEQ ID NO:15.

Additional methods for detecting or genotyping C. psittaci in a sampleinclude amplifying the nucleic acids in the sample with at least one C.psittaci and/or C. psittaci genotype specific primer by PCR, real-timePCR, RT-PCR, rt RT-PCR, ligase chain reaction or transcription mediatedamplification.

The disclosure also provides kits for detecting and/or genotyping C.psittaci in a sample suspected of containing a C. psittaci infection. Inone embodiment, the kit includes one or more primers as set forth as SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQID NO: 8. In another embodiment, the kit provides for the detection ofC. caviae in a sample, the kit including one of more primers set forthas SEQ ID NO: 16, or SEQ ID NO: 17.

I. Abbreviations cDNA complementary DNA ds double stranded DNAdeoxyribonucleic acid dNTP deoxyribonucleotides FAM carboxyfluoresceinFRET fluorescence resonance energy transfer HRM high-resolution melt JOE2,′7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein LLD lower limit ofdetection MEM minimal essential medium MOMP major outer membrane proteinompA outer membrane protein A PCR polymerase chain reaction RFLPrestriction fragment length polymorphism RT-PCR reverse transcriptasepolymerase chain reaction rt RT-PCR real-time reverse transcriptasepolymerase chain reaction RNA ribonucleic acid UTR untranslated regionsss single stranded TMA transcription-mediated amplificationII. Explanation Of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “a primer” includes single or pluralprimers and can be considered equivalent to the phrase “at least oneprimer.”

As used herein, the term “comprises” means “includes.” Thus, “comprisinga primer” means “including a primer” without excluding other elements.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for descriptivepurposes, unless otherwise indicated. Although many methods andmaterials similar or equivalent to those described herein can be used,particular suitable methods and materials are described below. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

To facilitate review of the various embodiments of the invention, thefollowing explanations of terms are provided:

Animal: A living multi-cellular vertebrate or invertebrate organism, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and veterinary subjects, such as birds or guineapigs.

Amplification: To increase the number of copies of a nucleic acidmolecule. The resulting amplification products are called “amplicons.”Amplification of a nucleic acid molecule (such as a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample. An example of amplification is thepolymerase chain reaction (PCR), in which a sample is contacted with apair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. This cycle can be repeated. The productof amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

Other examples of in vitro amplification techniques include quantitativereal-time PCR; reverse transcriptase PCR; real-time reversetranscriptase PCR (rt RT-PCR); nested PCR; strand displacementamplification (see U.S. Pat. No. 5,744,311); transcription-freeisothermal amplification (see U.S. Pat. No. 6,033,881, repair chainreaction amplification (see WO 90/01069); ligase chain reactionamplification (see EP-A-320 308); gap filling ligase chain reactionamplification (see U.S. Pat. No. 5,427,930); coupled ligase detectionand PCR (see U.S. Pat. No. 6,027,889); and NASBA™ RNA transcription-freeamplification (see U.S. Pat. No. 6,025,134) amongst others.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA alsocan contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA can besynthesized in the laboratory by reverse transcription from RNA.

Change: To become different in some way, for example to be altered, suchas increased or decreased. A detectable change is one that can bedetected, such as a change in the intensity, frequency or presence of anelectromagnetic signal, such as fluorescence. In some examples, thedetectable change is a reduction in fluorescence intensity. In someexamples, the detectable change is an increase in fluorescenceintensity. In some examples, the detectable change is a change inflorescence intensity as a result of a DNA melting curve of a testsample.

Chlamydophila abortus: Chlamydophila abortus (C. abortus) aregram-negative intracellular bacteria belonging to the Chlamydiaceaefamily. C. abortus is a species in Chlamydiae that causes abortion andfetal death in mammals, including humans. C. abortus was previouslyclassified as Chlamydophila psittaci along with all Chlamydiae exceptChlamydia trachomatis. This was based on a lack of evident glycogenproduction and on resistance to the antibiotic sulfadiazine. In 1999, C.psittaci and C. abortus were recognized as distinct species based ondifferences of pathogenicity and DNA-DNA reassociation.

C. abortus is endemic among ruminants and has been associated withabortion in a horse, a rabbit, guinea pigs, mice, pigs and humans.Infected females shed bacteria near the time of ovulation, so C. abortusis transmitted orally and sexually among mammals. All C. abortus strainswere isolated or PCR-amplified from placenta or fetal organs afterspontaneous abortion. C. abortus infection generally remains unapparentuntil an animal aborts late in gestation or gives birth to a weak ordead fetus. C. abortus has not been isolated from birds.

Chlamydophila caviae: Chlamydophila caviae (C. caviae) are gram-negativeintracellular bacteria belonging to the Chlamydiaceae family. C. caviaeis markedly specific for guinea pigs. Attempts to infect rabbits, mice,and hamsters have been unsuccessful. C. caviae infects primarily themucosal epithelium and is not invasive.

Chlamydophila psittaci: Chlamydophila psittaci(C. psittaci) aregram-negative intracellular bacteria belonging to the Chlamydiaceafamily. C. psittaci are classified on the basis of their genotypesdesignated A, B, C, D, E, F, and E/B in avian and two non-aviangenotypes (M56 (rodents) and WC (cattle)). Within these broadclassifications, the genotypes can be further characterized based ontheir serovars. Serovar A is endemic among psittacine birds and causeszoonotic disease in humans. Serovar B is endemic among pigeons, has beenisolated from turkeys, and can cause abortion in a dairy herd. Serovar Cisolates (GD, MT1, 91/1264, 91/1301, CT1 and Par1) were obtained from aGerman, Bulgarian and Belgian duck, a white swan, and a Californianturkey and a partridge, respectively. Serovar D has mainly been isolatedfrom turkeys but also from a seagull, a budgerigar, and from humans.Serovars C and D are known occupational hazards for poultry workers.Serovar E isolates, known as Cal-10, MP, or MN (meningopneumonitis),were isolated during an outbreak of human pneumonitis in the late 1920sand early 1930s. Subsequently, MN isolates have been obtained from avariety of birds worldwide, including ducks, pigeons, ostriches, andrheas. A single serovar F isolate, was obtained from a parakeet.

Direct detection of C. psittaci by cell culture is hazardous andrequires a level 3 laboratory, given its contagiousness. However, theinterpretation of serodiagnosis is difficult because of cross-reactionswith other species of Chlamydia and the high prevalence of Chlamydiapneumoniae in the general population.

Identification and genotyping of C. psittaci in avian samples andisolates is currently achieved by molecular methods such as outermembrane protein A (ompA) gene sequencing, restriction fragment lengthpolymorphism (RFLP), real-time PCR and microarray analysis. There areobvious limitations to these techniques as substantial amounts of a PCRamplicon are needed to produce distinctive and reproducible RFLPpatterns on ethidium bromide-stained agarose gels. Related genotypestend to have quite similar patterns, which may be difficult todistinguish, and typing results based on different enzyme patterns (e.g.AluI vs. MboII) may be contradictory. For example, C. psittaci isolateswere initially characterized by RFLP by Alul restriction mapping of themajor outer membrane protein gene ompl obtained after amplification bythe polymerase chain reaction. Digestion of C. psittaci ompl ampliconsby Alul generated several of the known distinct restriction patterns (A,B, D, E and F). However, restriction pattern C was not observed.Additionally, genetically aberrant strains cannot be genotyped using theabove-mentioned PCR-RFLP procedure. Sequencing of the ompA gene andalignment with type strain sequences can also be used to identify thegenotype of C. psittaci strains, since genotype-specific sites arelocated in the gene's variable domains (VD) VD2 and VD4, however thistechnique is time intensive. While the above techniques have improvedupon the traditional approaches to detect C. psittaci, they still lackspecificity and/or sensitivity to rapidly and accurately detect anddiscriminate C. psittaci genotypes in a biological sample.

C. psittaci 6BC ompA gene (ompA): The ompA-encoded gene of C. psittaci.As used herein “ompA” refers to the nucleotide sequence of ompA, thus aprobe or primer for ompA, such as those disclosed herein, capable ofhybridizing to the nucleotide sequence of ompA, such as the ompAnucleotide sequence given below (or the complement thereof).

An exemplary nucleotide sequence of C. psittaci 6BC ompA as found atGENBANK® Accession number X56980 on Mar. 13, 2009 is shown below:

(SEQ ID NO: 11)ttacactcttctacgagggtaattccaacttattctaagtggcataagaaataaaaatgtgtacaaaaatctgatagctcttttattagcaagtataaggagttattgcttgaaatctatgcctgaaaacagtcttttttcttatcgtctttactataataagaaaagtttgttatgttttcgaataatgaactgtatgttcatgcttaaggctgttttcacttgcaagacactcctcaaagccattaattgcctacaggatatcttgtctggctttaacttggacgtggtgccgccagaagagcaaattagaatagcgagcacaaaaagaaaagatactaagcataatctttagaggtgagtatgaaaaaactcttgaaatcggcattattgtttgccgctacgggttccgctctctccttacaagccttgcctgtagggaacccagctgaaccaagtttattaatcgatggcactatgtgggaaggtgcttcaggagatccttgcgatccttgcgctacttggtgtgacgccattagcatccgcgcaggatactacggagattatgttttcgatcgtgtattaaaagttgatgtgaataaaacttttagcggcatggctgcaactcctacgcaggctacaggtaacgcaagtaatactaatcagccagaagcaaatggcagaccgaacatcgcttacggaaggcatatgcaagatgcagagtggttttcaaatgcagccttcctagccttaaacatttgggatcgcttcgacattttctgcaccttaggggcatccaatggatacttcaaagcaagttcggctgcattcaacttggttgggttaatagggttttcagctgcaagctcaatctctaccgatcttccaatgcaacttcctaacgtaggcattacccaaggtgttgtggaattttatacagacacatcattttcttggagcgtaggtgcacgtggagctttatgggaatgtggttgtgcaactttaggagctgagttccaatacgctcaatctaatcctaagattgaaatgctcaacgtcacttcaagcccagcacaatttgtgattcacaaaccaagaggctataaaggagctagctcgaattttcctttacctataacggctggaacaacagaagctacagacaccaaatcagctacaattaaataccatgaatggcaagtaggcctcgccctgtcttacagattgaatatgcttgttccatatattggcgtaaactggtcaagagcaacttttgatgctgatactatccgcattgctcaacctaaattaaaatcggagattcttaacattactacatggaacccaagccttataggatcaaccactgctttgcccaataatagtggtaaggatgttctatctgatgtcttgcaaattgcttcgattcagatcaacaaaatgaagtctagaaaagcttgtggtgtagctgttggtgcaacgttaatcgacgctgacaaatggtcaatcactggtgaagcacgcttaatcaatgaaagagctgctcacatgaatgctcaattcagattctaaggatttagtttatactatcctaactttttaaaccgctatcagaacctgggagtctccgggttctgattttttaaataccacccttttc.

C. psittaci 90/105 ompA gene (ompA): The ompA-encoded gene of C.psittaci. As used herein “ompA” refers to the nucleotide sequence ofompA, thus a probe or primer for ompA, such as those disclosed herein,capable of hybridizing to the nucleotide sequence of ompA, such as theompA nucleotide sequence given below (or the complement thereof).

An exemplary nucleotide sequence of C. psittaci 90/105 ompA as found atGENBANK® Accession number AY762608 on Mar. 13, 2009 is shown below:

(SEQ ID NO: 12)atgaaaaaactcttgaaatcggcattattgtttgccgctacgggttccgctctctccttacaagccttgcctgtagggaacccagctgaaccaagtttattaatcgatggcactatgtgggaaggtgcttcaggagatccttgcgatccttgcgctacttggtgtgacgccattagcatccgcgcaggatactacggagattatgttttcgatcgtgtattaaaagttgatgtgaataaaacttttagcggcatggctgcaactcctacgcaggctacaggtaacgcaagtaatactaatcagccagaagcaaatggcagaccgaacatcgcttacggaaggcatatggaagatgcagagtggttttcaaatgcagccttcctagccttaaacatttgggatcgcttcgacattttctgcaccttaggggcatccaatggatacttcaaagcaagttcggctgcattcaacttggttgggttaatagggttttcagctgcaagctcaatctctaccgatcttccaacgcaacttcctaacgtaggcattacccaaggtgttgtggaattttatacagacacatcattttcttggagcgtaggtgcacgtggagctttatgggaatgtggttgtgcaactttaggagctgagttccaatacgctcaatctaatcctaagattgaaatgctcaacgtcacttcaagcccagcacaatttgtgattcacaaaccaagaggctataaaggagctagctcgaattttcctttacctataacggctggaacaacagaagctacagacaccaaatcagctacaattaaataccatgaatggcaagtaggcctcgccctgtcttacagattgaatatgcttgttccatatattggcgtaaactggtcaagagcaacttttgatgctgatactatccgcattgctcaacctaaattaaaatcggagattcttaacattactacatggaacccaagccttataggatcaaccactgctttgcccaataatagtggtaaggatgttctatctgatgtcttgcaaattgcttcgattcagatcaacaaaatgaagtctagaaaagcttgt 

C. psittaci 7778B15 ompA gene (ompA): The ompA-encoded gene of C.psittaci. As used herein “ompA” refers to the nucleotide sequence ofompA, thus a probe or primer for ompA, such as those disclosed herein,is capable of hybridizing to the nucleotide sequence of ompA, such asthe ompA nucleotide sequence given below (or the complement thereof). Anexemplary nucleotide sequence of C. psittaci 7778B15 ompA as found atGENBANK® Accession number AY762612 on Mar. 13, 2009 is shown below:

(SEQ ID NO: 13)atgaaaaaactcttgaaatcggcattattgtttgccgctacgggttccgctctctccttacaagccttgcctgtagggaacccagctgaaccaagtttattaatcgatggcactatgtgggaaggtgcttcaggagatccttgcgatccttgcgctacttggtgtgacgccattagcatccgcgcaggatactacggagattatgttttcgatcgtgtattaaaagttgatgtgaataaaactatcagcggtatgggtgcagctcctacaggaagcgcagcagccgattacaaaactcctacagatagacccaacatcgcttatggcaaacacttgcaagacgctgagtggttcacgaatgcagctttcctcgcattaaatatctgggatcgctttgatattttctgcacattaggtgcttccaatgggtacttcaaagctagttctgctgcattcaacctcgttggtttgattggtgttaaaggaacctccgtagcagctgatcaacttccaaacgtaggcatcactcaaggtattgttgagttttacacagatacaacattctcttggagcgtaggtgcacgtggtgctttatgggaatgtggttgtgcaactttaggagctgaattccagtatgctcaatctaatcctaaaattgaaatgctgaatgtaatctccagcccaacacaatttgtagttcacaagcctagaggatacaagggaacaggatcgaactttcctttacctctaacagctggtacagatggtgctacagatactaaatctgcaacactcaaatatcatgaatggcaagttggtttagcgctctcttacagattgaacatgcttgttccttacattggcgtaaactggtcaagagcaacttttgatgctgactctatccgcatcgctcaacctaaattagccgctgctgttttgaacttgaccacatggaacccaactcttttaggggaagctacagctttagatgctagcaacaaattctgcgacttcttacaaatcgcttcgattcagatcaacaaaatgaagtctagaaaagcttgt

Complementary: A double-stranded DNA or RNA strand consists of twocomplementary strands of base pairs. Complementary binding occurs whenthe base of one nucleic acid molecule forms a hydrogen bond to the baseof another nucleic acid molecule. Normally, the base adenine (A) iscomplementary to thymidine (T) and uracil (U), while cytosine (C) iscomplementary to guanine (G). For example, the sequence 5′-ATCG-3′ ofone ssDNA molecule can bond to 3′-TAGC-5′ of another ssDNA to form adsDNA. In this example, the sequence 5′-ATCG-3′ is the reversecomplement of 3′-TAGC-5′.

Nucleic acid molecules can be complementary to each other even withoutcomplete hydrogen-bonding of all bases of each molecule. For example,hybridization with a complementary nucleic acid sequence can occur underconditions of differing stringency in which a complement will bind atsome but not all nucleotide positions.

Consists essentially of: A transition phrase that limits the scope of aclaim to the specified materials or steps, and to those that do notmaterially affect the basic and novel characteristics of the claimedinvention. For example, a primer or probe that consists essentially of aspecified sequence can vary by an insignificant number of nucleotidesthat do not affect the overall sequence specificity of the primer orprobe, such as a variance by one, two, three or more nucleotides.

Detect: To determine if an agent (such as a signal or particularnucleotide or amino acid) is present or absent such as by detecting thepresence or absence of a detectable label. In some examples, this canfurther include quantification. For example, use of the disclosedprimers in particular examples permits detection of a fluorescentsignal, for example detection of a signal from a fluorogenic primer,which can be used to determine if a nucleic acid corresponding to anucleic acid of C. psittaci is present. Detection of a nucleic acid canbe direct as in the case of detection of a hybridized labeled primer orprobe. In other examples, detection of a nucleic acid is indirect as inthe case of hybridization of a primer or probe followed by amplificationand detection of a nucleic acid sequence.

Electromagnetic radiation: A series of electromagnetic waves that arepropagated by simultaneous periodic variations of electric and magneticfield intensity, and that includes radio waves, infrared, visible light,ultraviolet light, X-rays and gamma rays. In particular examples,electromagnetic radiation is emitted by a laser, which can possessproperties of monochromaticity, directionality, coherence, polarization,and intensity. Lasers are capable of emitting light at a particularwavelength (or across a relatively narrow range of wavelengths), forexample such that energy from the laser can excite a donor but not anacceptor fluorophore.

Emission or emission signal: The light of a particular wavelengthgenerated from a fluorophore after the fluorophore absorbs light at itsexcitation wavelengths.

Excitation or excitation signal: The light of a particular wavelengthnecessary to excite a fluorophore to a state such that the fluorophorewill emit a different (such as a longer) wavelength of light.

Fluorophore: A chemical compound, which when excited by exposure to aparticular stimulus such as a defined wavelength of light, emits light(fluoresces), for example at a different wavelength (such as a longerwavelength of light).

Fluorophores are part of the larger class of luminescent compounds.Luminescent compounds include chemiluminescent molecules, which do notrequire a particular wavelength of light to luminesce, but rather use achemical source of energy. Therefore, the use of chemiluminescentmolecules (such as aequorin) eliminates the need for an external sourceof electromagnetic radiation, such as a laser.

Examples of particular fluorophores that can be used in the primersand/or probes disclosed herein are known to those of skill in the artand include those provided in U.S. Pat. No. 5,866,366 to Nazarenko etal., such as 4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid;acridine and derivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide; BrilliantYellow; coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), ALEXA FLUOR®546, fluorescein, fluorescein isothiocyanate (FITC), QFITC (XRITC),-6-carboxy-fluorescein (HEX), and TET (Tetramethyl fluorescein);fluorescamine; IR144; IR1446; Malachite Green isothiocyanate;4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives such as pyrene, pyrene butyrate and succinimidyl1-pyrene butyrate; Reactive Red 4 (CIBACRON™. Brilliant Red 3B-A);rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA),tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC);sulforhodamine B; sulforhodamine 101 and sulfonyl chloride derivative ofsulforhodamine 101 (Texas Red); riboflavin; rosolic acid and terbiumchelate derivatives; LIGHTCYCLER® Red 640; Cy5.5; andCy56-carboxyfluorescein; boron dipyrromethene difluoride (BODIPY);acridine; stilbene; 6-carboxy-X-rhodamine (ROX); Texas Red; Cy3; Cy5,VIC® (Applied Biosystems); LIGHTCYCLER® Red 640; LIGHTCYCLER® Red 705;and Yakima yellow, amongst others.

Other suitable fluorophores include those known to those skilled in theart, for example those available from Invitrogen (Carlsbad, Calif.) orMOLECULAR PROBES® (Eugene, Oreg.). In particular examples, a fluorophoreis used as a donor fluorophore or as an acceptor fluorophore.

“Acceptor fluorophores” are fluorophores which absorb energy from adonor fluorophore, for example in the range of about 400 to 900 nm (suchas in the range of about 500 to 800 nm). Acceptor fluorophores generallyabsorb light at a wavelength which is usually at least 10 nm higher(such as at least 20 nm higher) than the maximum absorbance wavelengthof the donor fluorophore, and have a fluorescence emission maximum at awavelength ranging from about 400 to 900 nm. Acceptor fluorophores havean excitation spectrum which overlaps with the emission of the donorfluorophore, such that energy emitted by the donor can excite theacceptor. Ideally, an acceptor fluorophore is capable of being attachedto a nucleic acid molecule.

In a particular example, an acceptor fluorophore is a dark quencher,such as Dabcyl, QSY7™ (Molecular Probes), QSY33™ (Molecular Probes),BLACK HOLE QUENCHERS™ (Glen Research), ECLIPSE™ DARK QUENCHER™ (EpochBiosciences), or IOWA BLACK™ (Integrated DNA Technologies). A quenchercan reduce or quench the emission of a donor fluorophore. In such anexample, instead of detecting an increase in emission signal from theacceptor fluorophore when in sufficient proximity to the donorfluorophore (or detecting a decrease in emission signal from theacceptor fluorophore when a significant distance from the donorfluorophore), an increase in the emission signal from the donorfluorophore can be detected when the quencher is a significant distancefrom the donor fluorophore (or a decrease in emission signal from thedonor fluorophore when in sufficient proximity to the quencher acceptorfluorophore). In other examples, the fluorophore reaction can include aself-quenching moiety such as a hairpin configuration. In one suchexample, LUX™ (Invitrogen, Carlsbad, Calif.) primers can be used thatincorporate a fluorophore. The LUX™ primer technology uses one primerlabeled with a single fluorophore and containing a self-quenching moietyin conjunction with a corresponding unlabeled primer, bothcustom-synthesized according to the target nucleic acid of interest.Typically 15-40 bases in length, LUX™ primers are designed with afluorophore, such as FAM or JOE, near the 3′ end of the labeled primer.The 5′ end of the labeled primer includes a sequence that when in singlestranded conformation forms a hairpin structure. These properties of thelabeled primer intrinsically render it with fluorescence quenchingcapability, making a separate quenching moiety unnecessary. When thelabeled primer becomes incorporated into a double-stranded PCR product,the fluorophore is de-quenched, resulting in a significant increase influorescent signal (see FIG. 1).

“Donor Fluorophores” are fluorophores or luminescent molecules capableof transferring energy to an acceptor fluorophore, thereby generating adetectable fluorescent signal from the acceptor. Donor fluorophores aregenerally compounds that absorb in the range of about 300 to 900 nm, forexample about 350 to 800 nm. Donor fluorophores have a strong molarabsorbance coefficient at the desired excitation wavelength, for examplegreater than about 10³ M⁻¹ cm⁻¹.

Fluorescence Resonance Energy Transfer (FRET): A spectroscopic processby which energy is passed between an initially excited donor to anacceptor molecule separated by 10-100 Å. The donor molecules typicallyemit at shorter wavelengths that overlap with the absorption of theacceptor molecule. The efficiency of energy transfer is proportional tothe inverse sixth power of the distance (R) between the donor andacceptor (1/R⁶) fluorophores and occurs without emission of a photon. Inapplications using FRET, the donor and acceptor dyes are different, inwhich case FRET can be detected either by the appearance of sensitizedfluorescence of the acceptor or by quenching of donor fluorescence. Forexample, if the donor's fluorescence is quenched it indicates the donorand acceptor molecules are within the Förster radius (the distance whereFRET has 50% efficiency, about 20-60 Å), whereas if the donor fluorescesat its characteristic wavelength, it denotes that the distance betweenthe donor and acceptor molecules has increased beyond the Försterradius, such as when a TAQMAN® probe is degraded by Taq polymerasefollowing hybridization of the probe to a target nucleic acid sequenceor when a hairpin probe is hybridized to a target nucleic acid sequence.In another example, energy is transferred via FRET between two differentfluorophores such that the acceptor molecule can emit light at itscharacteristic wavelength, which is always longer than the emissionwavelength of the donor molecule.

Examples of oligonucleotides using FRET that can be used to detectamplicons include linear oligoprobes, such as HybProbes, 5′ nucleaseoligoprobes, such as TAQMAN® probes, hairpin oligoprobes, such asmolecular beacons, scorpion primers and UNIPRIMERS™, minor groovebinding probes, and self-fluorescing amplicons, such as sunrise primers.

High-Resolution Melt Analysis: High-resolution separation ofdouble-stranded nucleic acid material with heat (melting). Thetemperature at which a DNA strand separates and melts when heated canvary over a wide range, depending on the sequence, the length of thestrand, and the GC content of the strand. For example, meltingtemperatures can vary for products of the same length but differentGC/AT ratio, or for products with the same length and GC content, butwith a different GC distribution. Even a single base difference inheterozygous DNA can result in melting temperature shifts. Becausemelting temperatures vary according to these differences meltingtemperature profiles can be used to identify, distinguish and genotypeDNA products.

Conventional (standard) melt analysis is a fundamental property of DNAthat is often monitored with fluorescence. Conventional melting isperformed after Polymerase Chain Reaction (PCR) on any real-timeinstrument to monitor product purity (dsDNA dyes) and sequence(hybridization probes). Because PCR produces enough DNA for fluorescentmelting analysis, both amplification and analysis can be performed inthe same tube, thus providing a closed-tube system that requires noprocessing step, separation step or post-amplification manipulation.Dyes that stain double stranded DNA are commonly used to identifyproducts by their melting temperature (T_(m)). The T_(m) of a sample isdefined as the point at which half the probes have melted off the DNA.Alternatively, hybridization primers allow genotyping by melting ofproduct/primer duplexes.

The power of DNA melting analysis depends on its resolution. Recentadvances include high-resolution melt (HRM) analysis that providesuperior sensitivity and superiority between samples, such as allowing auser to perform mutation scanning of a sample. Conventional studies withultraviolet absorbance often require hours to collect high-resolutiondata at rates of 0.1-1.0° C./min to ensure equilibrium. In contrast,fluorescent melting analysis is usually acquired at 0.1-1.0° C./s,equilibrium is not achieved, and resolution is limited to 2-4 points/°C. In contrast, high-resolution melting can be performed rapidly with10-100 times the data density (50-100 points/° C.) of conventionalreal-time PCR instruments. HRM differs from conventional PCR productmelting T_(m) measurement in two ways. First, the accuracy of the meltcurve is maximized by acquiring fluorescence data over small temperatureincrements (as low as 0.01° C.). Second, the precise shape of the HRMcurve is a function of the DNA sequence being melted, allowing ampliconscontaining different sequences to be discriminated on the basis of meltcurve shape, irrespective of whether the amplicons share the same T_(m).HRM analysis makes use of melt curve normalization and comparisonsoftware that allows a user to determine whether two similar melt curvesdiffer from one another.

A melting temperature analysis can be performed on any instrument thatincludes a melt program. A melt program is usually performed afteramplification of the target nucleic acid, such as DNA. A typical meltprogram includes three segments:

(i) a segment that rapidly heats the sample to a temperature high enoughto denture all the DNA;

(ii) a segment that cools the samples to below the annealing temperatureof the target DNA; and

(iii) a segment that slowly heats the samples while measuring samplefluorescence as the target DNA melts.

The melting temperature analysis provides a melting curve of samplefluorescence versus temperature. For example, the chart may show adownward curve in fluorescence for the samples as they melt. Severalinstruments are commercially available that are capable of performingreal-time PCR and HRM analysis, for example, the ABI 7900 and 7900HTinstruments.

Hybridization: The ability of complementary single-stranded DNA or RNAto form a duplex molecule (also referred to as a hybridization complex).Nucleic acid hybridization techniques can be used to form hybridizationcomplexes between a primer (or probe) and a nucleic acid, such as a C.psittaci nucleic acid. For example, a primer (such as any of SEQ ID NOs:3-8) having some homology to a C. psittaci nucleic acid molecule willform a hybridization complex with a C. psittaci nucleic acid molecule(such as any of SEQ ID NOs: 11-13). Hybridization occurs between asingle stranded primer and a single stranded target nucleic acid (suchas a C. psittaci nucleic acid), as illustrated in FIG. 1. When thetarget nucleic acid is initially one strand of a duplex nucleic acid theduplex must be melted (at least partially) for the primer to hybridize.This situation is also illustrated in FIG. 1.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength (suchas the Na+ concentration) of the hybridization buffer will determine thestringency of hybridization. Calculations regarding hybridizationconditions for attaining particular degrees of stringency are discussedin Sambrook et al., (1989) Molecular Cloning, second edition, ColdSpring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). Thefollowing is an exemplary set of hybridization conditions and is notlimiting:

-   -   Very High Stringency (detects sequences that share at least 90%        identity)    -   Hybridization: 5×SSC at 65° C. for 16 hours    -   Wash twice: 2×SSC at room temperature (RT) for 15 minutes each    -   Wash twice: 0.5×SSC at 65° C. for 20 minutes each    -   High Stringency (detects sequences that share at least 80%        identity)    -   Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours    -   Wash twice: 2×SSC at RT for 5-20 minutes each    -   Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each    -   Low Stringency (detects sequences that share at least 50%        identity)    -   Hybridization: 6×SSC at RT to 55° C. for 16-20 hours    -   Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes        each.

The primers disclosed herein can hybridize to C. psittaci nucleic acidsunder low stringency, high stringency, and very high stringencyconditions. Generally, the primers hybridize to a C. psittaci nucleicacid under very high stringency conditions.

Isolated: An “isolated” biological component (such as a nucleic acid)has been substantially separated or purified away from other biologicalcomponents in which the component naturally occurs, such as otherchromosomal and extrachromosomal DNA, RNA, and proteins. Nucleic acidsthat have been “isolated” include nucleic acids purified by standardpurification methods. The term also embraces nucleic acids prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids, such as probes and primers. Isolated does not requireabsolute purity, and can include nucleic acid molecules that are atleast 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% oreven 100% isolated.

Label: An agent capable of detection, for example by spectrophotometry,flow cytometry, or microscopy. For example, a label can be attached to anucleotide, thereby permitting detection of the nucleotide, such asdetection of the nucleic acid molecule of which the nucleotide is apart. Examples of labels include, but are not limited to, radioactiveisotopes, enzyme substrates, co-factors, ligands, chemiluminescentagents, fluorophores, haptens, enzymes, and combinations thereof.Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed for example in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989)and Ausubel et al. (In Current Protocols in Molecular Biology, JohnWiley & Sons, New York, 1998).

LUX™ primers: FIG. 1 illustrates an oligonucleotide primer with areporter fluorophore, such as 6-carboxyfluorescein (FAM) or6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE) and a selfquenching moiety. In one embodiment, the LUX™ primer includes a 3′reporter fluorophore, such as FAM or JOE and a 5′ self quenching moiety,such as a hairpin structure. In the LUX™ primer, energy is released fromthe fluorophore in the form of light, upon extension of the primerduring PCR. LUX™ fluorogenic primers are generally produced as a pair oftwo primers. The first primer is labeled with a fluorophore, i.e. FAM(6-carboxyfluorescin), and the second primer is unlabeled. Due to thespecific conformation of the labeled primer (as a “hairpin” structure)prior to annealing to a target DNA sequence, interior fading of thefluorophore occurs (self-quenching). Annealing of the labeled primer tothe target DNA sequence results in extension of the labeled primer andleads to an enhancement of fluorophore fluorescence during PCR.

LUX™ primers can be used in real-time quantitative PCR and RT-PCR toquantify 100 or fewer copies of a target sequence (or gene) in as littleas 1 pg of template DNA or RNA. LUX™ primers have a broad dynamic rangeof 7-8 orders. For example, multiplex applications can be prepared usingseparate FAM and JOE-labeled primer sets to detect two different genesin the same sample. Typically, a custom-designed FAM-labeled primer setis used to detect the gene of interest, and a JOE-labeled Certified LUX™primer set is used to detect a housekeeping gene as an internal control.

LUX™ primers are compatible with a wide variety of real-time PCRinstruments, including but not limited to the ABI PRISM® 7700, 7000, and7900 and GeneAmp® 5700; the Bio-Rad iCycler™; the Stratagene Mx4000™ andMx3000™; the Cepheid SMART CYCLER®; the Corbett Research Rotor-Gene; andthe Roche LIGHTCYCLER®.

Nucleic acid (molecule or sequence): A deoxyribonucleotide orribonucleotide polymer including without limitation, cDNA, mRNA, genomicDNA, and synthetic (such as chemically synthesized) DNA or RNA. Thenucleic acid can be double stranded (ds) or single stranded (ss). Wheresingle stranded, the nucleic acid can be the sense strand or theantisense strand. Nucleic acids can include natural nucleotides (such asA, T/U, C, and G), and can also include analogs of natural nucleotides,such as labeled nucleotides. In one example, a nucleic acid is a C.psittaci nucleic acid, which can include nucleic acids purified from C.psittaci bacterium as well as the amplification products of such nucleicacids. A nucleic acid molecule for detection includes probes or primersthat are capable of hybridizing to the complementary sequence of atagert nucleic acid molecule of interest.

Nucleotide: The fundamental unit of nucleic acid molecules. A nucleotideincludes a nitrogen-containing base attached to a pentose monosaccharidewith one, two, or three phosphate groups attached by ester linkages tothe saccharide moiety.

The major nucleotides of DNA are deoxyadenosine 5′-triphosphate (dATP orA), deoxyguanosine 5′-triphosphate (dGTP or G), deoxycytidine5′-triphosphate (dCTP or C) and deoxythymidine 5′-triphosphate (dTTP orT). The major nucleotides of RNA are adenosine 5′-triphosphate (ATP orA), guanosine 5′-triphosphate (GTP or G), cytidine 5′-triphosphate (CTPor C) and uridine 5′-triphosphate (UTP or U).

Nucleotides include those nucleotides containing modified bases,modified sugar moieties and modified phosphate backbones, for example asdescribed in U.S. Pat. No. 5,866,336 to Nazarenko et al. (hereinincorporated by reference).

Examples of modified base moieties which can be used to modifynucleotides at any position on its structure include, but are notlimited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N˜6-sopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine amongstothers.

Examples of modified sugar moieties which may be used to modifynucleotides at any position on its structure include, but are notlimited to: arabinose, 2-fluoroarabinose, xylose, and hexose, or amodified component of the phosphate backbone, such as phosphorothioate,a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or aformacetal or analog thereof.

Polymerizing agent: A compound capable of reacting monomer molecules(such as nucleotides) together in a chemical reaction to form linearchains or a three-dimensional network of polymer chains. A particularexample of a polymerizing agent is polymerase, an enzyme which catalyzesthe 5′ to 3′ elongation of a primer strand complementary to a nucleicacid template. Examples of polymerases that can be used to amplify anucleic acid molecule include, but are not limited to the E. coli DNApolymerase I, specifically the Klenow fragment which has 3′ to 5′exonuclease activity, Taq polymerase, reverse transcriptase (such ashuman immunodeficiency virus-1 reverse transcriptase (HIV-1 RT)), E.coli RNA polymerase, and wheat germ RNA polymerase II.

The choice of polymerase is dependent on the nucleic acid to beamplified. If the template is a single-stranded DNA molecule, aDNA-directed DNA or RNA polymerase can be used; if the template is asingle-stranded RNA molecule, then a reverse transcriptase (such as anRNA-directed DNA polymerase) can be used.

Probes and Primers: Nucleic acid molecules for detection. Nucleic acidprobes and primers can be readily prepared based on the nucleic acidmolecules provided in this invention, and therefore provide asubstantial utility for the disclosed sequences. A probe comprises anisolated nucleic acid capable of hybridizing to a complementary sequenceof a target nucleic acid (such as a portion of a C. psittaci nucleicacid), and a detectable label or reporter molecule can be attached to aprobe. A primer comprises a short nucleic acid molecule, such as a DNAoligonucleotide, for example sequences of at least 15 nucleotides, whichcan be annealed to a complementary target nucleic acid molecule bynucleic acid hybridization to form a hybrid complex between the primerand the target nucleic acid strand. A primer can be extended along thetarget nucleic acid molecule by a polymerase enzyme such as a PCRtechnique. Therefore, primers can be used to amplify a target nucleicacid molecule (such as a portion of a C. psittaci nucleic acid), whereinthe sequence of the primer is specific for the target nucleic acidmolecule, for example so that the primer will hybridize to the targetnucleic acid molecule under very high stringency hybridizationconditions.

The specificity of a probe or primer increases with its length. Thus,for example, a primer that includes 30 consecutive nucleotides willanneal to a target sequence with a higher specificity than acorresponding primer of only 15 nucleotides. Thus, to obtain greaterspecificity, primers can be selected that include at least 15, 20, 22,24, 26, 28, 30, 35, 40 or more consecutive nucleotides.

In particular examples, a probe or primer is at least 15 contiguousnucleotides complementary to a target nucleic acid molecule. Particularlengths of probes or primers that can be used to practice the methods ofthe present disclosure (for example, to amplify a region of a C.psittaci nucleic acid) include probes or primers having at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40 or more contiguous nucleotidescomplementary to the target nucleic acid molecule (to be detected byhybridization or to be amplified), such as a primer of 15-30nucleotides, 15-40 nucleotides.

A “set of primers” is a group of more than one primer and can be as fewas a two primers, or a “primer pair.” Primer pairs can be used foramplification of a nucleic acid sequence, for example, by PCR, real-timePCR, or other nucleic-acid amplification methods known in the art. An“upstream” or “forward” primer is a primer 5′ to a reference point on anucleic acid sequence. A “downstream” or “reverse” primer is a primer 3′to a reference point on a nucleic acid sequence. In general, at leastone forward and one reverse primer are included in an amplificationreaction. PCR primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, ® 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.).

Methods for preparing and using probes and primers are described in, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y.; Ausubel et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences.

In particular examples, a probe of primer (or primer pair) includes adetectable label. A detectable label or reporter molecule can beattached to a primer. Typical labels include radioactive isotopes,enzyme substrates, co-factors, ligands, chemiluminescent or fluorescentagents, haptens, and enzymes.

Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley-Intersciences (1987).

In one example, a probe or primer includes at least one fluorophore,such as an acceptor fluorophore or donor fluorophore. For example, afluorophore can be attached at the 5′- or 3′-end of the primer. Inspecific examples, the fluorophore is attached to the base at the 5′-endof the primer, the base at its 3′-end, the phosphate group at its 5′-endor a modified base, such as a thymidine internal to the primer. In oneexample, a primer pair includes a single-labeled fluorogenic primer anda corresponding unlabeled primer, such as a D-LUX™ or LUX™ primer pair.In a further embodiment, the labeled primer includes a5′-carboxyfluorescin (FAM) labeled primer and a self-quenching moiety,such as a hairpin structure.

Quantitating a nucleic acid molecule: Determining or measuring aquantity (such as a relative quantity) of nucleic acid moleculespresent, such as the number of amplicons or the number of nucleic acidmolecules present in a sample. In particular examples, it is determiningthe relative amount or actual number of nucleic acid molecules presentin a sample.

Quenching of fluorescence: A reduction of fluorescence. For example,quenching of a fluorophore's fluorescence occurs when a quenchermolecule (such as the fluorescence quenchers listed above) is present insufficient proximity to the fluorophore that it reduces the fluorescencesignal (for example, prior to the binding of a primer to a C. psittacinucleic acid sequence, when the primer contains a fluorophore and aself-quenching moiety).

Real-Time PCR: A method for detecting and measuring products generatedduring each cycle of a PCR, which are proportionate to the amount oftemplate nucleic acid prior to the start of PCR. The informationobtained, such as an amplification curve, can be used to determine thepresence of a target nucleic acid (such as a C. psittaci nucleic acid)and/or quantitate the initial amounts of a target nucleic acid sequence.In some examples, real-time PCR is real-time reverse transcriptase PCR(rt RT-PCR).

In some examples, the amount of amplified target nucleic acid (such as aC. psittaci nucleic acid) is detected using a labeled probe, such as aprobe labeled with a fluorophore, for example a TAQMAN® probe. In thisexample, the increase in fluorescence emission is measured in real-time,during the course of the real-time PCR. This increase in fluorescenceemission is directly related to the increase in target nucleic acidamplification (such as C. psittaci nucleic acid amplification). In someexamples, the change in fluorescence (dRn) is calculated using theequation dRn=Rn⁺−Rn⁻, with Rn⁺ being the fluorescence emission of theproduct at each time point and Rn⁻ being the fluorescence emission ofthe baseline. The dRn values are plotted against cycle number, resultingin amplification plots for each sample

In another example, the amount and identification of an amplified targetnucleic acid (such as a C. psittaci nucleic acid) is detected using alabeled primer, such as a primer labeled with a fluorophore, for examplea LUX™ primer (Invitrogen, CA). In this example, an increase influorescence emission is observed and measured upon exposure of thesample to real-time PCR conditions as the labeled primer transitionsfrom a single stranded conformation to a double stranded product/primerduplex. An increase in fluorescence emission is directly related tohybridization of the labeled primer to the target nucleic acidamplification product (such as C. psittaci nucleic acid amplificationproducts) and the characteristics of the amplification product, forexample, G/C content, and sequence length. Conversely, a decrease influorescence emission of the amplified nucleic acids upon incrementaltemperature increases as part of a melt curve analysis is the result ofthe labeled primer separating from the amplification product, andtransitioning back to a single-stranded conformation, where minimalfluorescence is observed as a result of self-quenching of thefluorophore moiety. With reference to FIGS. 3A-3C and 5A-5B, thethreshold value is the PCR cycle number at which the fluorescenceemission (dRn) exceeds a chosen threshold (Rn⁻), which is typically 10times the standard deviation of the baseline (this threshold level can,however, be changed if desired).

Sample: A sample, such as a biological sample, is a sample obtained froman animal subject. As used herein, biological samples include allclinical samples useful for detection of C. psittaci infection insubjects, including, but not limited to, cells, tissues, and bodilyfluids, such as: blood; derivatives and fractions of blood, such asserum; extracted galls; biopsied or surgically removed tissue, includingtissues that are, for example, unfixed, frozen, fixed in formalin and/orembedded in paraffin; tears; milk; skin scrapes; surface washings;urine; sputum; cerebrospinal fluid; prostate fluid; pus; bone marrowaspirates; bronchoalveolar levage; tracheal aspirates; sputum;nasopharyngeal aspirates; pharyngeal swabs, oropharyngeal aspirates; andsaliva. In particular embodiments, the biological sample is obtainedfrom an animal subject, such as in the form of tracheal aspirates,sputum, nasopharyngeal aspirates, pharyngeal swabs, oropharyngealaspirates, and saliva.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N8O5, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn, and tblastx. Blastn is used tocompare nucleic acid sequences, while blastp is used to compare aminoacid sequences. Additional information can be found at the NCBI website.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresent in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1554 nucleotidesis 75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 15 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(i.e., 15÷20*100=75).

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions. Stringent conditions are sequence-dependent and aredifferent under different environmental parameters.

The primers disclosed herein are not limited to the exact sequencesshown, as those skilled in the art will appreciate that changes can bemade to a sequence, and not substantially affect the ability of theprimer to function as desired. For example, sequences having at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to any of SEQ ID NOS:3-8 and SEQID NOS:16-17 are provided herein. One of skill in the art willappreciate that these sequence identity ranges are provided for guidanceonly; it is possible that primers can be used that fall outside theseranges.

Signal: A detectable change or impulse in a physical property thatprovides information. In the context of the disclosed methods, examplesinclude electromagnetic signals such as light, for example light of aparticular quantity or wavelength. In certain examples, the signal isthe disappearance of a physical event, such as quenching of light.

Target nucleic acid molecule: A nucleic acid molecule whose detection,quantitation, qualitative detection, or a combination thereof, isintended. The nucleic acid molecule need not be in a purified form.Various other nucleic acid molecules can also be present with the targetnucleic acid molecule. For example, the target nucleic acid molecule canbe a specific nucleic acid molecule (which can include RNA such as viralRNA), the amplification of which is intended. Purification or isolationof the target nucleic acid molecule, if needed, can be conducted bymethods known to those in the art, such as by using a commerciallyavailable purification kit or the like. In one example, a target nucleicmolecule is a chlamydophila nucleic acid sequence. In another example,the chlamydophila nucleic sequence is a C. psittaci, C. caviae or C.abortus nucleic acid. In several examples, a target nucleic molecule isa C. psittaci nucleic acid sequence.

III. Overview Of Several Embodiments

Outbreaks of psittacosis in poultry farms and zoonotic infections inworkers who are in close proximity with infected birds raise publichealth concerns. Additionally, the importation of companion birdsinfected with virulent bacterial infections poses an increased risk ofinfection for individuals involved with the sale, transportation andownership of these animals. Methods are needed to readily detect andidentify C. psittaci, for example to rapidly diagnose or determine thepotential of bacteria samples, such as those obtained from a subjectinfected or believed to be infected with C. psittaci. Additionally, itwould be particularly advantageous to be able to detect and discriminatebetween Chlamydophila species.

Disclosed herein are methods for the universal detection of C. psittacias well as for the identification of C. psittaci genotypes. Furthermore,the methods allow for the discrimination between closely relatedChlamydophila species such as C. abortus and C. caviae. The methods havebeen developed in one embodiment with a unique set of nucleic acidprimers that are surprisingly effective at detecting and discriminatingbetween Chlamydophila species. In another embodiment, the nucleic acidprimers are surprisingly effective at detecting and discriminatingbetween genotypes of C. psittaci. This ability to rapidly screen andidentify a Chlamydophila species or C. psittaci genotype provides asignificant public health advantage.

In particular examples, the methods for the detection and identificationof Chlamydophila involve direct dection of a hybridized primer or probe,such as by Southern blot or dot blot analysis. In other examples,hybridized primers or probes are further used to direct amplification ofa target Chlamydophila nucleic acid, which is then detected using alabel such as a self-quenching fluororophore.

As disclosed herein, using sequence alignments of Chlamydophila and C.psittaci sequences, previously unknown regions of high sequencinghomology were discovered amongst individual Chlamydophila species andstrains. These regions were used to create the primers shown in Table 1.Using these highly homologous regions as a starting point the disclosedprimers were designed such that they were surprisingly effective atrecognizing genetically similar isolates within Chlamydophila and withinC. psittaci genotypes. In an effort to reduce false positive reactionsamongst Chlamydophila species, sets of primers were designed thatallowed for the specific amplification of C. psittaci nucleic acids. Ina further development, primer sets were designed that allowed for thespecific amplification of an individual C. psittaci genotype.Additionally, elimination of false positive reactions among similargenetic strains of Chlamydophila was achieved using primer sets thatallowed for the specific amplification of C. caviae nucleic acids. Thelatter primer set was surprisingly effective at recognizing andidentifying C. caviae nucleic acids in a sample.

Primers and Probes

Primers and probes that can hybridize to and direct the amplification ofChlamydophila target nucleic acids are disclosed. The primers and probesdisclosed herein are between 15 to 40 nucleotides in length, such as 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or even 40 nucleotides in length. In severalembodiments, the primer or probe is capable of hybridizing under veryhigh stringency conditions to a complementary sequence of aChlamydophila nucleic acid sequence set forth as SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and directingthe amplification of the Chlamydophila nucleic acid. In someembodiments, the primer or probe is capable of hybridizing under veryhigh stringency conditions to the complementary sequence of a C.psittaci nucleic acid sequence set forth as SEQ ID NO: 11, SEQ ID NO:12, or SEQ ID NO: 13, and directing the amplification of the C. psittacinucleic acid. In another embodiment, the primer is capable ofhybridizing under very high stringency conditions to the complementarysequence of a C. caviae nucleic acid sequence set forth as SEQ ID NO: 14and directing the amplification of the C. caviae nucleic acid. In yetanother embodiment, the primer is capable of hybridizing under very highstringency conditions to the complementary sequence of a C. abortusnucleic acid sequence set forth as SEQ ID NO: 15 and directing theamplification of the C. abortus nucleic acid.

In several embodiments, the primer or probe capable of hybridizing toand directing the amplification of a C. psittaci nucleic acid is 15 to40 nucleotides in length and includes a nucleic acid sequence that is atleast 95% identical such as at least 96%, at least 97%, at least 98%, atleast 99%, or even 100% identical to the nucleic acid sequence set forthas SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,or SEQ ID NO: 8. In several embodiments, the primer capable ofhybridizing to and directing the amplification of a C. psittaci nucleicacid consists essentially of, or consists of a nucleic acid sequence setforth as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, or SEQ ID NO: 8.

In one embodiment, the primer is capable of hybridizing under very highstringency conditions to and directing the amplification of a C. caviaenucleic acid contains a nucleic acid sequence that is at least 95%identical such as at least 96%, at least 97%, at least 98%, at least99%, or even 100% identical to the nucleic acid sequence set forth asSEQ ID NO: 16, or SEQ ID NO: 17. In another embodiment, the primercapable of hybridizing to and directing the amplification of a C. caviaenucleic acid consists essentially of, or consists of a nucleic acidsequence set forth as SEQ ID NO: 16, or SEQ ID NO: 17.

In several embodiments, the primer is a C. psittaci genotype-specificprimer. In one example, a C. psittaci genotype-specific primer iscapable of hybridizing under stringent conditions (such as highstringency, or very high stringency conditions) to a complementary C.psittaci nucleic acid from a specific genotype, such as C. psittacigenotype A, B, C, D, E or F. In one embodiment, a primer that is C.psittaci genotype-specific for C. psittaci genotype F is not specificfor the amplification and hybridization of any other C. psittacigenotype. Likewise, a primer that is genotype-specific for theamplification and hybridization of C. psittaci genotype A is notspecific for the amplification and hybridization of C. psittaci genotypeF. In other words, in the above two instances, a nucleic acid primerthat specifically hybridizes to a C. psittaci genotype F nucleic acid(such as a nucleic acid that is at least a portion of the ompA gene fromC. psittaci, for example the nucleic acid sequence set forth as SEQ IDNOs: 11-13) does not amplify and hybridize to nucleic acids of anotherC. psittaci genotype. Conversely, a nucleic acid primer thatspecifically hybridizes to C. psittaci genotype E and/or B nucleic acidsdoes not specifically hybridize to C. psittaci genotype F nucleic acids;such nucleic acids would be genotype-specific primers for C. psittacigenotypes B and/or E. Thus, in some embodiments, genotype-specificprimers can be used to specifically amplify a nucleic acid from C.psittaci genotype F or from C. psittaci genotypes E/B, but not both.

In some embodiments, the primer is capable of hybridizing under veryhigh stringency conditions to a complementary nucleic acid fromChlamydophila, for example the amplification and hybridization of a C.caviae nucleic acid from the ompA gene of C. caviae set forth as SEQ IDNO: 14. In yet another embodiment, the primer is capable of hybridizingunder very high stringency conditions to a complentary nucleic acid fromChlamydophila, for example the amplification and hybridization of a C.abortus nucleic acid from the ompA gene of C. abortus set forth as SEQID NO: 15.

In several embodiments, the primer is capable of hybridizing andamplifying under very high stringency conditions to one or more C.psittaci genotypes. In one example, a C. psittaci specific primer iscapable of hybridizing under stringent conditions (such as highstringency, or very high stringency conditions) to the complementarysequence of any C. psittaci nucleic acid, for example, genotypes A, B,C, D, E, F or E/B. For example, a primer specific for the amplificationand hybridization of C. psittaci can detect any C. psittaci genotype andis not limited to the detection of a single C. psittaci genotype. Inother words, a nucleic acid primer that specifically hybridizes to acomplementary sequence of a C. psittaci nucleic acid (such as a nucleicacid that is at least a portion of the ompA gene from C. psittaci suchas SEQ ID NOs: 11-13) does not hybridize to a C. caviae or C. abortusnucleic acid; such primers would be specific for the detection of C.psittaci.

In some embodiments, the primer is capable of distinguishing between C.psittaci genotypes. In some embodiments, the primer specific for thehybridization and amplification of a C. psittaci nucleic acid includes anucleic acid sequence at least 95% identical such as at least 96%, atleast 97%, at least 98%, at least 99%, or even 100% identical to SEQ IDNO:3 or SEQ ID NO: 4. In a specific example, the primer thatdiscriminates between C. psittaci genotypes contains a nucleic acidsequence at least 95% identical such as at least 96%, at least 97%, atleast 98%, at least 99%, or even 100% identical to SEQ ID NO: 5 or SEQID NO: 6. In another embodiment, a primer capable of hybridizing undervery high stringency conditions to C. psittaci genotype F includes anucleic acid sequence at least 95% identical such as at least 96%, atleast 97%, at least 98%, at least 99%, or even 100% identical to SEQ IDNO: 7 or SEQ ID NO: 8.

In some embodiments, the primer is specific for the amplification of C.psittaci genotypes A, B, C, D or E, such as the nucleic acid sequenceset forth as SEQ ID NO: 5 or SEQ ID NO: 6. In a specific example, aprimer specific for C. psittaci genotypes A, B, C, D or E includes anucleic acid sequence at least 95% identical such as at least 96%, atleast 97%, at least 98%, at least 99%, or even 100% identical to SEQ IDNO:5 or SEQ ID NO: 6. In another embodiment, a primer specific for C.psittaci genotypes A, B, C, D or E consists essentially of, or consistsof a nucleic acid set forth as SEQ ID NO:5 or SEQ ID NO: 6. In someexamples, the primer is specific for the amplification of C. psittacigenotype F, such as the nucleic acid sequence set forth as SEQ ID NO: 7or SEQ ID NO: 8. In a specific example, a primer specific for theamplification of C. psittaci genotype F includes a nucleic acid sequenceat least 95% identical such as at least 96%, at least 97%, at least 98%,at least 99%, or even 100% identical to SEQ ID NO: 7 or SEQ ID NO: 8. Inanother embodiment, a primer specific for C. psittaci genotype Fconsists essentially of a nucleic acid set forth as SEQ ID NO: 7 or SEQID NO: 8.

In certain embodiments the primers are a set of primers, such as a pairof primers, capable of hybridizing to and amplifying a Chlamydophilanucleic acid. Such a set of primers includes at least one forward primerand at least one reverse primer, where the primers are specific for theamplification of a Chlamydophila nucleic acid in a sample. In someexamples, the set of primers includes a pair of primers that is specificfor the amplification of C. psittaci, C. caviae or C. abortus.

In certain examples, the pair of primers is specific for theamplification of a C. psittaci nucleic acid and includes a forwardprimer at least 95% identical such as at least 96%, at least 97%, atleast 98%, at least 99%, or even 100% identical to SEQ ID NO: 5 and areverse primer at least 95% identical such as at least 96%, at least97%, at least 98%, at least 99%, or even 100% identical to SEQ ID NO: 6.In another example, the pair of primers specific for the amplificationof a C. caviae nucleic acid includes a forward primer at least 95%identical such as at least 96%, at least 97%, at least 98%, at least99%, or even 100% identical to SEQ ID NO: 17 and a reverse primer atleast 95% identical such as at least 96%, at least 97%, at least 98%, atleast 99%, or even 100% identical to SEQ ID NO: 16.

In another example, a set of primers specific for the amplification of aC. psittaci nucleic acid includes one or more forward primers 15 to 40nucleotides in length including a nucleic acid sequence at least 95%identical such as at least 96%, at least 97%, at least 98%, at least99%, or even 100% identical to SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:7; and one or more reverse primers 15 to 40 nucleotides in lengthincluding a nucleic acid sequence at least 95% identical such as atleast 96%, at least 97%, at least 98%, at least 99%, or even 100%identical to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In oneexample, the pair of primers is specific for the amplification ofnucleic acids from at least one C. psittaci genotype. In anotherexample, the set of primers is specific for the amplification of nucleicacids from at least one C. psittaci genotype selected from A, B, C, D, Eor F. In a further embodiment, the set of primers is specific for theamplification of C. psittaci genotypes A, B, C, and E. In yet anotherembodiment, the set of primers is specific for the amplification of C.psittaci genotype F nucleic acids.

In yet another example, a set of primers specific for the amplificationof a C. caviae nucleic acid includes one or more forward primers 15 to40 nucleotides in length including a nucleic acid sequence at least 95%identical such as at least 96%, at least 97%, at least 98%, at least99%, or even 100% identical to SEQ ID NO: 17 and one or more reverseprimers 15 to 40 nucleotides in length including a nucleic acid sequenceat least 95% identical such as at least 96%, at least 97%, at least 98%,at least 99%, or even 100% identical to SEQ ID NO: 16.

Although exemplary primers are provided in SEQ ID NOs: 3-10, one skilledin the art will appreciate that the primer sequence can be variedslightly by moving the primers a few nucleotides upstream or downstreamfrom the nucleotide positions that they hybridize to on the C. psittacinucleic acid, provided that the primer is still specific for the C.psittaci sequence, meaning that the primer retains species- orstrain-specificity for C. psittaci. For example, the primer is specificfor the hybridization to a complementary sequence of a C. psittacinucleic acid set forth as SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:13. In another example, one of skill in the art will appreciate that byanalyzing the consensus sequences shown in FIGS. 4A and 4B thatvariations of the primers disclosed as SEQ ID NOs: 3-8 can be made by“sliding” the primers a few nucleotides 5′ or 3′ from their positions,and that such variation will still be specific for C. psittaci. Thus, insome examples, the primer sequence is 2-5 nucleotides 5′ of SEQ ID NOs:3-8 and/or 2-5 nucleotides 3′ if SEQ ID NOs: 3-8 using the sequencespresented in FIGS. 4A and 4B. Thus, the primers can be 2, 3, 4 or 5nucleotides 5′ of SEQ ID NOs: 3-8 and/or 2, 3, 4 or 5 nucleotides 3′ ifSEQ ID NOs: 3-8.

Also provided by the present application are primers that includevariations to the nucleotide sequences shown in any of SEQ ID NOs: 3-8,as long as such variations permit detection of the C. psittaci nucleicacid, such as a C. psittaci genotype. For example, a primer can have atleast 95% sequence identity such as at least 96%, at least 97%, at least98%, at least 99% to a nucleic acid containing the sequence shown in anyof SEQ ID NOs: 3-8. In such examples, the number of nucleotides does notnecessarily change, but the nucleic acid sequence shown in any of SEQ IDNOs: 3-8 can vary at a few nucleotides, such as changes at 1, 2, 3, or 4nucleotides, for example by changing the nucleotides as shown in theprimer binding location of FIGS. 4A and 4B.

The present application also provides primers that are slightly longeror shorter than the nucleotide sequences shown in any of SEQ ID NOs:3-8, as long as such deletions or additions permit detection of thedesired C. psittaci nucleic acid, such as specific C. psittacigenotypes. For example, a primer can include a few nucleotide deletionsor additions at the 5′- or 3′-end of the primer shown in any of SEQ IDNOs: 3-8, such as addition or deletion of 1, 2, 3, or 4 nucleotides fromthe 5′- or 3′-end, or combinations thereof (such as a deletion from oneend and an addition to the other end). In such examples, the number ofnucleotides changes. One of skill in the art will appreciate that theconsensus sequences shown in FIGS. 4A and 4B provide sufficient guidanceas to what additions and/or subtractions can be made, while stillmaintaining specificity for the detection of C. psittaci and/or C.psittaci genotype nucleic acids.

In several embodiments, the primer is detectably labeled, either with anisotopic or non-isotopic label, alternatively the target nucleic acid(such as a C. psittaci nucleic acid) is labeled. Non-isotopic labelscan, for instance, comprise a fluorescent or luminescent molecule,biotin, an enzyme or enzyme substrate or a chemical. Such labels arepreferentially chosen such that the hybridization of the primer withtarget nucleic acid (such as a C. psittaci nucleic acid) can bedetected. In some examples, the primer is labeled with a fluorophore.Examples of suitable fluorophore labels are given above. In someexamples, the fluorophore is a donor fluorophore. In other examples, thefluorophore is an accepter fluorophore, such as a fluorescence quencher.In some examples, the primer includes both a donor fluorophore and anaccepter fluorophore. In other examples, the primer includes afluorophore and a self quenching moiety. In one example, the primerincludes a fluorophore on a modified nucleotide (such as a T within theprimer), and the labeled primer further includes a self-quenchingmoiety, such as a hairpin structure. Appropriate donor/acceptorfluorophore pairs can be selected using routine methods. In one example,the donor emission wavelength is one that can significantly excite theacceptor, thereby generating a detectable emission from the acceptor.

In particular examples, the self quenching moiety (a fluorescencequencher) is attached to the 5′ end of the primer and the donorfluorophore is attached to a 3′ end of the primer. In another particularexample, the self quenching moiety (such as a fluorescence quencher) isattached to the 3′ end of the primer and the donor fluorophore isattached to the 5′ end of the primer.

Detection and Identification of Chlamydophila and Chlamydophila psittaci

The Chlamydophila and C. psittaci specific primers disclosed herein canbe used for the detection, identification and genotyping ofChlamydophila and C. psittaci in a sample, such as a biological sampleobtained from a subject that has or is suspected of having aChlamydophila and/or C. psittaci infection. Thus, the disclosed methodscan be used to diagnose if a subject has a Chlamydophila and/or a C.psittaci infection and/or discriminate between the bacterial speciesand/or strain the subject is infected with. An example of the methods ofidentifying Chlamydophila species is shown in FIG. 6A.

In particular examples, the methods for the detection and identificationof Chlamydophila involve direct dection of a hybridized primer or probe,such as by Southern blot or dot blot analysis. In other examples,hybridized primers or probes are further used to direct amplification ofa target Chlamydophila nucleic acid, which is then detected using alabel such as a self-quenching fluororophore. In particular examples,amplification is carried out usings pairs of the particular primersdisclosed herein. In other examples, amplification is carried our usinga specific forward or reverse primer disclosed herein, in combinationwith a universal reverse or forward primer (respectively). Any universalprimer known to the art that will hybridize to a common repeat sequenceand direct the amplification of DNA will be suitable for the methodsdescribed herein. In particular examples, the nucleic acid sample forthe subject is pretreated to add a repeat nucleotide sequence that canserve as a target sequence for a universal amplification primer.

In one embodiment, a method for diagnosing a Chlamydophila psittaciinfection in a subject suspected of having a C. psittaci infectionincludes obtaining a nucleic acid sample from the subject and contactingthe sample with one or more of the probes or primers specific for C.psittaci as disclosed herein (such as SEQ ID NOs: 3-8), and detectinghybridization or amplification of the one or more C. psittaci specificprimers in the sample. Detection of hybridization or amplificationindicates that the subject is infected with C. psittaci. The amplifiednucleic acid can be detected by a detectable label, such as afluorescent moiety conjugated to one the amplification primers. Inanother embodiment, the method further includes discriminating and/ordistinguishing between a C. psittaci infection and a C. caviae infectionby contacting the sample suspected of having a C. psittaci or C. caviaeinfection with one or more primers set forth as SEQ ID NO: 16 or SEQ IDNO: 17, and detecting hybridization to or amplification of the sample.Detection of hybridization or amplification indicates that the subjectis infected with C. caviae. In yet another embodiment, the methodfurther includes discriminating and/or distinguishing between a C.psittaci infection and a C. abortus infection by contacting the samplesuspected of having a C. psittaci or C. abortus infection with one ormore primers set forth as SEQ ID NO: 5 or SEQ ID NO: 6, and detectinghybridization to or amplification of the sample. Detection ofhybridization or amplification indicates that the subject is infectedwith C. psittaci. The absence of hybridization or amplificationindicates that the subject is infected with C. abortus.

Methods for the detection of Chlamydophila nucleic acids are disclosed,for example to determine if a subject is infected with Chlamydophilabacteria. Methods also are provided for determining the species and/orgenotype of the Chlamydophila nucleic acid, for example to determinewhich species and/or genotype of Chlamydophila bacteria a subject isinfected with.

The methods described herein may be used for any purpose for whichdetection of Chlamydophila or C. psittaci is desirable, includingdiagnostic and prognostic applications, such as in laboratory andclinical settings. Appropriate samples include any conventionalenvironmental or biological samples, including clinical samples obtainedfrom a human or veterinary subject, such as a bird or mammal. Suitablesamples include all biological samples useful for detection of bacterialinfection in subjects, including, but not limited to, cells, tissues(for example, lung, liver and kidney), bone marrow aspirates, bodilyfluids (for example, blood, serum, urine, cerebrospinal fluid,bronchoalveolar levage, tracheal aspirates, sputum, nasopharyngealaspirates, oropharyngeal aspirates, saliva), eye swabs, cervical swabs,vaginal swabs, rectal swabs, stool, and stool suspensions. Particularlysuitable samples include samples obtained from bronchoalveolar levage,tracheal aspirates, sputum, nasopharyngeal aspirates, oropharyngealaspirates, or saliva. Standard techniques for acquisition of suchsamples are available. See for example, Schluger et al., J. Exp. Med.176:1327-1333, 1992; Bigby et al., Am. Rev. Respir. Dis. 133:515-518,1986; Kovacs et al., NEJM 318:589-593, 1988; and Ognibene et al., Am.Rev. Respir. Dis. 129:929-932, 1984.

In some embodiments, detecting a Chlamydophila nucleic acid in a sampleinvolves contacting the sample with at least one of the Chlamydophilaspecific primers or probes disclosed herein that is capable ofhybridizing to a complementary Chlamydophila nucleic acid underconditions of very high stringency (such as a nucleic acid primer orprobe capable of hybridizing under very high stringency conditions to acomplementary sequence of a Chlamydophila nucleic acid sequence setforth as SEQ ID NOs: 11-15; for example a primer or probe with a nucleicacid sequence at least 95% identical, such as at least 96%, at least97%, at least 98%, at least 99%, or even 100% identical to thenucleotide sequence set forth as one of SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, andSEQ ID NO: 17), and detecting hybridization between the sample and theprimer or probe. Detection of hybridization between the primer or probeand the sample indicates the presence of a Chlamydophila nucleic acid inthe sample.

By using Chlamydophila species-specific primers or probes, the disclosedmethods can be used to detect the presence of Chlamydophila species in asample. For example, by contacting the sample with a C. psittacispecies-specific primer or probe as disclosed herein, such as a primeror probe capable of hybridizing under very high stringency conditions toa complementary sequence of a C. psittaci nucleic acid sequence setforth as SEQ ID NOs: 11-13; for example, a primer with a nucleic acidsequence of at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6.Detection of hybridization of the C. psittaci species-specific primer orprobe to the sample indicates the presence of C. psittaci nucleic acidsin the sample.

In another embodiment, Chlamydophila species-specific primers can beused to discriminate between species of Chlamydophila in a sample. Forexample, by contacting the sample with the Chlamydophilaspecies-specific primer or probe as disclosed herein, such as a primeror probe capable of hybridizing under very high stringency conditions toa complementary sequence of a C. psittaci nucleic acid, a C. abortusnucleic acid, or a C. caviae nucleic acid; for example, a primer with anucleic acid sequence of at least 95% identical to SEQ ID NO: 5, SEQ IDNO: 6 (primers specific for C. psittaci and C. caviae but not C.abortus) or SEQ ID NO: 16, SEQ ID NO: 17 (primers specific for C.caviae). Detection of hybridization of the Chlamydophilaspecies-specific primers (SEQ ID NO: 5 or SEQ ID NO: 6) to the sampleindicates the presence of Chlamydophila nucleic acids in the sample.Furthermore, hybridization between primers SEQ ID NO: 16 or SEQ ID NO:17 is indicative of the presence of C. caviae in the sample.Alternatively, the absence of hybridization in the sample with either ofthe above primer sets (SEQ ID NO: 5/SEQ ID NO: 6 or SEQ ID NO: 16/SEQ IDNO: 17) is indicative of the absence of C. psittaci and C. caviae in thesample. However, detection of hybridization or amplification of SEQ IDNOs: 3 or 4 but not SEQ ID NOs: 5 and 6 is indicative of a C. abortusinfection.

Additionally, the primers or probes disclosed herein can be used todetect the presence of and discriminate between genotypes of C.psittaci. For example, contacting a sample with a primer or probespecific for C. psittaci genotype F nucleic acids, such as a primer orprobe capable of hybridizing under very high stringency conditions to acomplementary sequence of a C. psittaci genotype F nucleic acid such asthe sequence set forth as SEQ ID NOs: 11-13, for example a primer orprobe with a nucleic acid sequence of at least 95% identical to SEQ IDNO: 7 or SEQ ID NO: 8 can be used to detect C. psittaci genotype Fnucleic acids in the sample. Detection of hybridization between the C.psittaci genotype F-specific primer or probe and the sample indicatesthe presence of C. psittaci genotype F nucleic acids in the sample.Thus, the disclosed methods can be used discriminate between genotypesof C. psittaci in a sample.

In another example, a primer specific for C. psittaci genotype A, B, C,D or E, such as a primer or probe capable of hybridizing under very highstringency conditions to a complementary sequence of a C. psittacinucleic acid sequence set forth as SEQ ID NOs: 11-13, for example anucleic acid set forth as SEQ ID NO: 5 or SEQ ID NO: 6, may be used todetect hybridization between the primer or probe and a sample suspectedto contain C. psittaci. Detection of hybridization is indicative that atleast one genotype from C. psittaci genotype A, B, C, D, E, or F ispresent in the sample. In one embodiment, the primer or probe includes anucleic acid sequence at least 95% identical, such as at least 96%, atleast 97%, at least 98%, at least 99%, or even 100% identical to thenucleotide sequence set forth as SEQ ID NO: 5 or SEQ ID NO: 6.

In yet another example, contacting a sample with a primer or probespecific for C. psittaci genotype F, such as a primer or probe capableof hybridizing under very high stringency conditions to a complementarysequence of a C. psittaci nucleic acid sequence set forth as SEQ ID NOs:11-13, for example a nucleic acid set forth as SEQ ID NO: 7 or SEQ IDNO: 8, and detecting the hybridization between the primer or probe andthe C. psittaci nucleic acid indicates the presence of C. psittacigenotype F. In one embodiment, the primer or probe includes a nucleicacid sequence at least 95% identical, such as at least 96%, at least97%, at least 98%, at least 99%, or even 100% identical to thenucleotide sequence set forth as SEQ ID NO: 7 or SEQ ID NO: 8. In afurther embodiment, the primer or probe consists essentially of thenucleotide sequence set forth as SEQ ID NO: 7 or SEQ ID NO: 8.

In one example, contacting a sample with a primer or probe specific forC. caviae, such as a primer capable of hybridizing under very highstringency conditions to a complementary sequence of a C. caviae nucleicacid sequence set forth as SEQ ID NO: 14, for example a nucleic acid setforth as SEQ ID NO: 16 or SEQ ID NO: 17, and detecting the hybridizationbetween the primer and the sample suspected of containing a C. caviaenucleic acid indicates the presence of C. caviae. In one embodiment, theprimer or probe specific for C. caviae includes a nucleic acid sequenceat least 95% identical to the nucleotide sequence set forth as SEQ IDNO: 16 or SEQ ID NO: 17. In another embodiment, the primer or probespecific for C. caviae consists essentially of the nucleotide sequenceset forth as SEQ ID NO: 16 or SEQ ID NO: 17.

In yet another example (and as shown in FIG. 6A), a primer or probespecific for the detection of C. abortus or C. psittaci can be used todistinguish between these two Chlamydophila species in a biologicalsample. In this example, a primer or probe capable of hybridizing undervery high stringency conditions, for example a nucleic acid set forth asSEQ ID NO: 3 or SEQ ID NO: 4, is used to detect hybridization betweenthe primer or probe and the sample suspected of containing aChlamydophila nucleic acid. Detection of hybridization and amplificationof the sample by the primers or probes set forth as SEQ ID NO: 3 or SEQID NO: 4 suggests the presence of C. psittaci, C. abortus, or C. caviaein the sample. In this example, the presence of C. abortus is confirmedby detecting a lack of hybridization and amplification using the primersset forth as SEQ ID NO: 5 or SEQ ID NO: 6 and the sample. A failure todetect hybridization or amplification is indicative of the presence ofC. abortus in the sample. In contrast, detection of amplification usingSEQ ID NO: 5 or SEQ ID NO: 6 in the above example is indicative of thepresence of C. psittaci. Additional C. psittaci strain-specific primersmay be used to determine the genotype of C. psittaci as alreadydescribed. Following amplication with SEQ ID NO: 3 or SEQ ID NO: 4 orSEQ ID NO: 5 or SEQ ID NO: 6, HRM analysis can then be performed todistinguish between the presence of C. psittaci and C. caviae in thesample

In some embodiments, detecting the presence of a Chlamydophila or a C.psittaci nucleic acid sequence in a sample includes the extraction ofChlamydophila and/or C. psittaci RNA. RNA extraction relates toreleasing RNA from a latent or inaccessible form in an environment suchas a virion, cell or sample and allowing the RNA to become freelyavailable. In such a state, it is suitable for effective detectionand/or amplification of the Chlamydophila and/or C. psittaci nucleicacid. Releasing RNA may include steps that achieve the disruption ofbacterial cells containing RNA. Extraction of RNA is generally carriedout under conditions that effectively exclude or inhibit anyribonuclease activity that may be present. Additionally, extraction ofRNA may include steps that achieve at least a partial separation of theRNA dissolved in an aqueous medium from other cellular or viralcomponents, wherein such components may be either particulate ordissolved.

One of ordinary skill in the art will know suitable methods forextracting RNA from a sample; such methods will depend upon, forexample, the type of sample in which the Chlamydophila and C. psittaciRNA is found. For example, the RNA may be extracted using guanidiniumisothiocyanate, such as the single-step isolation by acid guanidiniumisothiocyanate-phenol-chloroform extraction of Chomczynski et al., Anal.Biochem. 162:156-59, 1987. The sample can be used directly or can beprocessed, such as by adding solvents, preservatives, buffers, or othercompounds or substances. Bacterial RNA can be extracted using standardmethods. For instance, rapid RNA preparation can be performed using acommercially available kit (such as the SIGMA-ALDRICH® GenEluteBacterial Total RNA Purification Kit, All-In-One Purification Kit(NORGEN BIOTEC CORPORATION), TRIzol® MAX™ Bacterial RNA Isolation Kit(Invitrogen™), MESSEGEAMP™ II-Bacteria RNA Amplification Kit (AMBION®)or RNEASY® Protect Bacteria Mini Kit (QIAGEN). Alternatively,Chlamydophila bacteria may be disrupted by a suitable detergent in thepresence of proteases and/or inhibitors of ribonuclease activity.

In some embodiments, the primer is detectably labeled, either with anisotopic or non-isotopic label; in alternative embodiments, theChlamydophila nucleic acid is labeled. Non-isotopic labels can, forinstance, comprise a fluorescent or luminescent molecule, or an enzyme,co-factor, enzyme substrate, or hapten. The primer is incubated with asingle-stranded or double-stranded preparation of RNA, DNA, or a mixtureof both, and hybridization determined. In some examples, hybridizationresults in a detectable change in signal such as in increase or decreasein signal, for example from the labeled primer. Thus, detectinghybridization includes detecting a change in signal from the labeledprimer during hybridization relative to signal from the labeled primerbefore hybridization. In another embodiment, detecting hybridizationincludes detecting a change in signal from the labeled primer afterhybridization relative to signal from the labeled primer beforehybridization. In some examples, detecting hybridization furtherincludes performing high-resolution melt analysis of an amplifiedproduct in a sample and determining the signal from the labeled primerrelative to signal from the labeled primer before amplification. Inother instances, detecting hybridization includes performinghigh-resolution melt analysis of an amplified product in a sample anddetermining a change in signal from the labeled primer during thehigh-resolution melt analysis. As already discussed, a change inconformation of the labeled primer (such as a LUX™ primer) fromsingle-stranded conformation to double-stranded conformation, forexample, as a result of amplification will result in an increase offluorescence. Conversely, a shift in conformation of the labeled primer(such as a LUX™ primer) from double-stranded to single-strandedconformation or a hairpin structure will result in a significantdecrease in fluorescence, and will therefore be detected as a decreasein signal. One of ordinary skill in the art will appreciate that othermeans for detecting hybridization are commercially available and arereadily applicable to the proposed methods. While the method fordetecting hybridization is not critically important, the ability todetect a change in signal relative to the initial starting material(e.g., labeled primer alone) is necessary.

In some embodiments, Chlamydophila nucleic acids present in a sample areamplified prior to using a hybridization primer or probe for detection.For instance, it can be advantageous to amplify a portion of theChlamydophila or C. psittaci nucleic acid, then detect the presence ofthe amplified Chlamydophila or C. psittaci nucleic acid. For example, toincrease the number of nucleic acids that can be detected, therebyincreasing the signal obtained. Chlamydophila or C. psittaci specificnucleic acid primers can be used to amplify a region that is at leastabout 50, at least about 60, at least about 70, at least about 80, atleast about 90, at least about 100, at least about 200, or more basepairs in length to produce amplified Chlamydophila or C. psittacinucleic acids. Any nucleic acid amplification method can be used todetect the presence of Chlamydophila or C. psittaci in a sample. In onespecific, non-limiting example, polymerase chain reaction (PCR) is usedto amplify the Chlamydophila or C. psittaci nucleic acid sequences. Inother specific, non-limiting examples, real-time PCR, reversetranscriptase-polymerase chain reaction (RT-PCR), real-time reversetranscriptase-polymerase chain reaction (rt RT-PCR), ligase chainreaction, or transcription-mediated amplification (TMA) is used toamplify the Chlamydophila or C. psittaci nucleic acid. In a specificexample, the Chlamydophila or C. psittaci nucleic acid is amplified byrt RT-PCR. Techniques for nucleic acid amplification are well-known tothose of skill in the art.

Typically, at least two primers are utilized in the amplificationreaction, however it is envisioned that one primer can be utilized, forexample to reverse transcribe a single stranded nucleic acid such as asingle-stranded Chlamydophila or C. psittaci RNA, followed byhybridization with a Chlamydophila or C. psittaci primer or probe.

Amplification of a Chlamydophila or a C. psittaci nucleic acid involvescontacting the Chlamydophila or C. psittaci nucleic acid with one ormore primers that are capable of hybridizing to and directing theamplification of the Chlamydophila or C. psittaci nucleic acid (such asa nucleic acid capable of hybridizing under very high stringencyconditions to the complementary Chlamydophila or C. psittaci nucleicacid). In one embodiment, amplification of a Chlamydophila or a C.psittaci nucleic acid involves contacting the Chlamydophila or C.psittaci nucleic acid with one or more primers that are capable ofhybridizing to and directing the amplification of the Chlamydophila orC. psittaci nucleic acid, such as a nucleic acid capable of hybridizingunder very high stringency conditions to a complementary sequence of aChlamydophila or C. psittaci nucleic acid set forth as SEQ NOs: 11-15,for example a primer that is 15-40 nucleotides long and is at least 95%identical, such as at least 96%, at least 97%, at least 98%, at least99%, or even 100% identical to the nucleotide sequence set forth as SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the sampleis contacted with at least one primer that is specific for a C. psittacigenotype, such as those disclosed herein.

In some embodiments, the sample is contacted with at least one pair ofprimers that include a forward and reverse primer that both hybridize toa Chlamydophila nucleic acid specific for C. psittaci and/or a C.psittaci genotype, such as C. psittaci genotype A, B, C, D, E, or F.Examples of suitable primer pairs for the amplification of Chlamydophilaand/or C. psittaci and/or C. psittaci genotype-specific nucleic acidsare described above.

In one example, the sample is contacted with a pair of primers capableof hybridizing to and amplifying a complementary sequence of a C.psittaci nucleic acid including at least one forward primer 15-40nucleotides long that is at least 95% identical to the nucleotidesequence set forth as SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, andat least one reverse primer 15-40 nucleotides longs that is at least 95%identical, such as at least 96%, at least 97%, at least 98%, at least99%, or even 100% identical to the nucleotide sequence set forth as SEQID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In another example, the forwardand reverse primers include multiple forward and reverse primers thatare specific for Chlamydophila psittaci and include nucleic acidsequences 15-40 nucleotides longs that are at least 95% identical, suchas at least 96%, at least 97%, at least 98%, at least 99%, or even 100%identical to the nucleotide sequences set forth as SEQ ID NOs: 3-8. Inyet another example, the forward and reverse primers are specific forChlamydophila psittaci genotypes A, B, C, D or E, and include a nucleicacid sequence at 15-40 nucleotides longs that is least 95% identical,such as at least 96%, at least 97%, at least 98%, at least 99%, or even100% identical to the nucleotide sequences set forth as SEQ ID NO: 5 orSEQ ID NO: 6. In one embodiment, primers specific for the amplificationof Chlamydophila psittaci genotype F include a nucleic acid sequence at15-40 nucleotides longs that is least 95% identical to the nucleotidesequence set forth as SEQ ID NO: 7 or SEQ ID NO: 8.

In instances where hybridization results in a detectable change insignal, for example from the labeled primer, hybridization can bedetermined for example by high-resolution melt analysis of the amplifiedPCR product. In this example, the amplified PCR product is subjected tohigh temperature, for example between 70° C. and 85° C., resulting inseparation of the double-stranded amplified PCR product into asingle-stranded conformation. For example, a LUX™ primer that hybridizesto nucleic acids in a sample can be extended during amplification, forexample using PCR techniques, to form a double-stranded amplificationproduct; this in turn can be separated into a single-strandedamplification product upon exposure to high temperature, therebyresulting in a decrease in fluorescence of the LUX™ primer label. Insome instances, high-resolution melt analysis is performed on theamplified product at a temperature of about 75° C.-85° C. in 0.1° C.increments. In another embodiment, high-resolution melt analysis isperformed on the amplified product at a temperature of about 75° C.-85°C. in 0.05° C. increments. In yet another embodiment, thehigh-resolution melt analysis includes two normalization regions, thefirst between about 75° C. and about 77° C., before separation of theamplified product, and a second between about 83° C. and 85° C., afterseparation of the amplified product. The inclusion of normalizationregions is particularly useful when comparing unrelated samples. Theincorporation of normalization regions into the high-resolution meltanalysis allows a user to eliminate variations in the melt analysis thatmay occur as a result of an artifact in the test system.

An example of the differentiation of C. psittaci genotypes is shown inFIG. 6B. In one embodiment, the amplification of sample nucleic acids bySEQ ID NOs: 5 and 6, followed by HRM analysis of the amplified productscan be used to identify the presence of C. psittaci genotypes A-F or C.caviae in the sample. In such embodiments, distinct melt curves areestablished herein to determine the presence of C. psittaci genotypes A,B, C, E as well as C. caviae. The melt curves of C. psittaci genotypes Dand F are indistinguishable. Thus, in particular examples, followingdetection of the distinct genotype D/F curve in a sample, nucleic acidamplification with or hybridization of the primers set forth as SEQ IDNOs: 7 and 8 is necessary to differentiate between genotype D andgenotype F. The ability to amplify or detect nucleic acids in the samplewith the primers set forth as SEQ ID NOs: 7 and 8 is indicative of C.psittaci genotype F. The absence of amplification or detection isindicative of C. psittaci genotype D.

Any type of thermal cycler apparatus can be used for the amplificationof the Chlamydophila and C. psittaci nucleic acids and/or thedetermination of hybridization. Examples of suitable apparatuses includea PTC-100® Peltier Thermal Cycler (MJ Research, Inc.; San Francisco,Calif.), a ROBOCYCLER® 40 Temperature Cycler (Stratagene; La Jolla,Calif.), or a GENEAMP® PCR System 9700 (Applied Biosystems; Foster City,Calif.). For real-time PCR, any type of real-time thermocycler apparatuscan be used. For example, a ROTOR-GENE Q®, (QIAGEN; Germantown, Md.), aBioRad ICYCLER IQ™, LIGHTCYCLER™ (Roche; Mannheim, Germany), a 7700SEQUENCE DETECTOR® (Perkin Elmer/Applied Biosystems; Foster City,Calif.), ABI™ systems such as the 7000, 7500, 7700, or 7900 systems(Applied Biosystems; Foster City, Calif.), or an MX4000™, MX3000™ orMX3005™ (Stratagene; La Jolla, Calif.), and Cepheid SMARTCYCLER™ can beused to amplify nucleic acid sequences in real-time.

The amplified Chlamydophila or C. psittaci nucleic acid, for example aChlamydophila species or C. psittaci genotype specific nucleic acid, canbe detected in real-time, for example by real-time PCR such as real-timeRT-PCR, in order to determine the presence, the identity, and/or theamount of a Chlamydophila or C. psittaci genotype specific nucleic acidin a sample. In this manner, an amplified nucleic acid sequence, such asan amplified Chlamydophila or C. psittaci nucleic acid sequence, can bedetected using a primer specific for the product amplified from theChlamydophila or C. psittaci sequence of interest, such as any of theChlamydophila primers or probes that are specific for C. psittacigenotypes A, B, C, D, E, or F discussed herein. Detecting the amplifiedproduct includes the use of labeled primers that are sufficientlycomplementary and hybridize to the nucleic acid sequence of interest,whereupon the primers are extended during PCR amplification. Thus, thepresence, amount, and/or identity of the amplified product can bedetected by hybridizing a labeled primer, such as a fluorescentlylabeled primer, complementary to the amplified product. In oneembodiment, the detection of a nucleic acid sequence of interestincludes the combined use of PCR amplification and a labeled primer suchthat the product is measured using real-time RT-PCR. In anotherembodiment, the detection of an amplified nucleic acid sequence ofinterest includes high-resolution melt analysis of the amplified nucleicacid sequence, such as a melt curve, for example a melt curve withnormalization regions that separate the amplified product under hightemperature and record the change in fluorescence of the labeled primeras compared to the level of fluorescence of the amplified product priorto high-resolution melt analysis. In yet another embodiment, thedetection of an amplified nucleic acid sequence of interest includes thehybridization and amplification of the nucleic acid to primers disclosedherein and separation of the labeled primer and amplified product underhigh-resolution melt analysis, where a shift in fluorescence as comparedto the amplified product indicates a change in conformation of thelabeled primer. In some embodiments, detection of the change in signalfrom the labeled primer occurs after amplification of the sample. Inanother embodiment, detection of the change in signal from the labeledprimer occurs after high-resolution melt analysis of the sample.

In one embodiment, the fluorescently-labeled primers rely uponfluorescence resonance energy transfer (FRET), or in a change in thefluorescence emission wavelength of a sample, as a method to detecthybridization of a DNA primer to the amplified target nucleic acid inreal-time. For example, FRET that occurs between fluorogenic labels ondifferent probes or primers (for example, using HYBPROBES®) or between afluorophore and a non-fluorescent quencher on the same probe or primer(for example, using a molecular beacon, LUX™ primer or a TAQMAN® probe)can identify a probe or primer that specifically hybridizes to the DNAsequence of interest and in this way, using Chlamydophila or C. psittacispecific probes or primers, can detect the presence, identity, and/oramount of a Chlamydophila or a C. psittaci in a sample. In oneembodiment, the fluorescently-labeled DNA primers used to identifyamplification products have spectrally distinct emission wavelengths,thus allowing them to be distinguished within the same reaction tube.

In another embodiment, a melting curve analysis of the amplified targetnucleic acid can be performed subsequent to the amplification process.The T_(m) of a nucleic acid sequence depends on, for example, the lengthof the sequence, its G/C content and its G/C distribution. Thus, theidentification of the T_(m) for a nucleic acid sequence can be used toidentify the amplified nucleic acid.

Kits

The nucleic acid primers disclosed herein can be supplied in the form ofa kit for use in the detection, identification, and/or genotyping ofChlamydophila or C. psittaci. In several embodiments, the nucleic acidprimers disclosed herein discriminate between Chlamydophila species. Inanother example, the nucleic acid primers disclosed herein distinguishbetween strains of C. psittaci. In yet another embodiment, the nucleicacid primers disclosed discriminate between a C. psittaci and a C.caviae nucleic acid. In yet another embodiment, the nucleic acid primersdisclosed herein discriminate between a C. psittaci and a C. abortusnucleic acid. In some embodiments, the nucleic acid primers disclosedherein discriminate between genotypes of C. psittaci.

The nucleic acid primers disclosed herein can be supplied in the form ofa kit for use in the detection, identification, and/or genotyping ofChlamydophila or C. psittaci in a sample. In such a kit, an appropriateamount of one or more of the nucleic acid primers disclosed herein isprovided in one or more containers or held on a substrate. A nucleicacid primer may be provided suspended in an aqueous solution or as afreeze-dried or lyophilized powder, for instance. The container(s) inwhich the nucleic acid(s) are supplied can be any conventional containerthat is capable of holding the supplied form, for instance, microfugetubes, ampoules, or bottles. The kits can include either labeled orunlabeled nucleic acid primers for use in detection, identification, andgenotyping of Chlamydophila or C. psittaci nucleotide sequences.

In some applications, one or more primers (as described above), such aspairs of primers, may be provided in pre-measured single use amounts inindividual, typically disposable, tubes or equivalent containers. Withsuch an arrangement, the sample to be tested for the presence ofChlamydophila or C. psittaci nucleic acids can be added to theindividual tubes and amplification carried out directly. In oneembodiment, hybridization of the primers to nucleic acids in a sample isdetermined by PCR techniques. In another embodiment, hybridization ofthe primers to nucleic acids in a sample is determined byhigh-resolution melt analysis of the amplified PCR product. In someexamples, high-resolution melt analysis is performed on the solution inthe same tube in which the sample was amplified. One advantage of theabove system is the ability to amplify, screen and detect amplificationof nucleic acids, such as Chlamydophila or C. psittaci nucleic acids,within a single reaction vessel, thereby reducing the likelihood ofcontamination.

The amount of nucleic acid primer supplied in the kit can be anyappropriate amount, and may depend on the target market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each nucleic acid primer provided wouldlikely be an amount sufficient to prime several PCR amplificationreactions. General guidelines for determining appropriate amounts may befound in Innis et al., Sambrook et al., and Ausubel et al. A kit mayinclude more than two primers in order to facilitate the PCRamplification of a larger number of Chlamydophila or C. psittacinucleotide sequences in a single test reaction.

In some embodiments, kits also may include the reagents necessary tocarry out PCR amplification reactions, including DNA sample preparationreagents, appropriate buffers (such as polymerase buffer), salts (forexample, magnesium chloride), and deoxyribonucleotides (dNTPs).

One or more control sequences for use in the PCR reactions also may besupplied in the kit (for example, for the detection of human RNAse P).

Particular embodiments include a kit for detecting and genotyping aChlamydophila or C. psittaci nucleic acid based on the componentsdescribed above. Such a kit includes at least one primer specific for aChlamydophila or C. psittaci nucleic acid (as described herein) andinstructions. A kit may contain more than one different primer, such as2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50, 100, or moreprimers. The instructions may include directions for obtaining a sample,processing the sample, preparing the primers, and/or contacting eachprimer with an aliquot of the sample. In certain embodiments, the kitincludes an apparatus for separating the different primers, such asindividual containers (for example, microtubules) or an array substrate(such as, a 96-well or 384-well microtiter plate). In particularembodiments, the kit includes prepackaged primers, such as primerssuspended in suitable medium in individual containers (for example,individually sealed EPPENDORF® tubes) or the wells of an array substrate(for example, a 96-well microtiter plate sealed with a protectiveplastic film). In other particular embodiments, the kit includesequipment, reagents, and instructions for extracting and/or purifyingnucleotides from a sample both prior to, and after, amplification.

Synthesis of Oligonucleotide Primers

In vitro methods for the synthesis of oligonucleotides are well known tothose of ordinary skill in the art; such methods can be used to produceprimers for the disclosed methods. The most common method for in vitrooligonucleotide synthesis is the phosphoramidite method, formulated byLetsinger and further developed by Caruthers et al. (Methods Enzymol.154:287-313, 1987). This is a non-aqueous, solid phase reaction carriedout in a stepwise manner, wherein a single nucleotide (or modifiednucleotide) is added to a growing oligonucleotide. The individualnucleotides are added in the form of reactive 3′-phosphoramiditederivatives. See also, Gait (Ed.), Oligonucleotide Synthesis. Apractical approach, IRL Press, 1984.

In general, the synthesis reactions proceed as follows: Adimethoxytrityl or equivalent protecting group at the 5′ end of thegrowing oligonucleotide chain is removed by acid treatment. (The growingchain is anchored by its 3′ end to a solid support such as a siliconbead.) The newly liberated 5′ end of the oligonucleotide chain iscoupled to the 3′-phosphoramidite derivative of the next deoxynucleotideto be added to the chain, using the coupling agent tetrazole. Thecoupling reaction usually proceeds at an efficiency of approximately99%; any remaining unreacted 5′ ends are capped by acetylation so as toblock extension in subsequent couplings. Finally, the phosphite triestergroup produced by the coupling step is oxidized to the phosphotriester,yielding a chain that has been lengthened by one nucleotide residue.This process is repeated, adding one residue per cycle. See, forexample, U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 4,973,679, and5,132,418. Oligonucleotide synthesizers that employ this or similarmethods are available commercially (for example, the PolyPlexoligonucleotide synthesizer from Gene Machines, San Carlos, Calif.). Inaddition, many companies will perform such synthesis (for example,Sigma-Genosys, The Woodlands, Tex.; Qiagen Operon, Alameda, Calif.;Integrated DNA Technologies, Coralville, Iowa; and TriLinkBioTechnologies, San Diego, Calif.).

The following examples are provided to illustrate particular features ofcertain embodiments. However, the particular features described belowshould not be construed as limitations on the scope of the invention,but rather as examples from which equivalents will be recognized bythose of ordinary skill in the art.

EXAMPLE 1 Materials and Methods

This example describes the materials and methods used to determine thespecificity and sensitivity of the disclosed primers to detect anddiscriminate between species of Chlamydophila and strains of C.psittaci.

Bacterial Strains and Specimens

The disclosed primers were tested for specificity of C. psittaci nucleicacids using reference strain isolates DD-34 (ATCC VR-854), CP3 (ATCCVR-574), CT1, NJ1, MN (ATCC Vr-122), and VS-225 along with 169 specimensacquired from companion or aviary birds and mammals. The specimens werepreviously submitted to the Infectious Diseases Laboratory at theCollege of Veterinary Medicine, University of Georgia, and testedpositive at the time of collection (2004 to 2007) for C. psittaci orother Chlamydophila species by a PCR-based assay. All specimens wereobtained from a recommended specimen source: i.e., conjunctival,choanal, or cloacal swabs or whole blood.

C. psittaci Culture

C. psittaci reference strains were propagated in Vero cell monolayersgrown in 25−cm² culture flasks in Eagle's minimal essential medium (MEM)supplemented with MEM nonessential amino acids, 2 μM L-glutamine, 20 μMHEPES buffer, 10% fetal calf serum, 20 μg/ml streptomycin, and 25 μg/mlvancomycin. Confluent Vero cell monolayers were inoculated by replacingthe growth medium with 1 ml of stock C. psittaci culture diluted 1:10 inMEM containing 1 μg/ml of cycloheximide. The inoculated monolayers wereplaced at 37° C. for 2 hours before an additional 4 ml of MEM containingcycloheximide was added to each flask. Cultures were incubated for 7days at 37° C. or until the monolayers demonstrated approximately 70%cytopathic effect and the remaining Vero cells were scraped from theflask into the medium. One milliliter of each culture was centrifuged at20,000×g for 60 min, and the pellet was resuspended in nuclease-freewater (Promega Co.) and used for DNA extraction using standardconditions. The remaining culture was dispensed into aliquots and frozenat −70° C. Titration of cultures was performed with 96-well flat-bottommicrotiter plates containing Vero cells. Frozen C. psittaci cultures of50 μl quantities at 10-fold dilutions were used to inoculate wells ofVero cells in triplicate. After incubation for 72 hours at 37° C. in a5% CO₂ atmosphere, the medium was removed and the cells were fixed withmethanol and stained with a Chlamydia genus-specific monoclonal antibody(Bio-Rad). Inclusions (inclusion forming units/ml) were counted using aninverted fluorescence microscope.

DNA Extraction for Real-Time PCR

DNA from C. psittaci cultures was extracted using a QiaAmp DNA minikit(Qiagen, Inc.) according to the manufacturer's instructions. The DNA waseluted into 200 μl of Qiagen elution buffer and stored at −70° C. untiltested.

LUX™ Primer Design and Optimization

Primer sets targeting the variable regions of the C. psittaci ompA genewere designed. LUX™ chemistry (Invitrogen, CA) utilizes a5-carboxyfluorescein (FAM)-labeled primer and a corresponding unlabeledprimer. All primer sets were designed using the C. psittaci 6BC ompAgene (GENBANK® Accession no. X56980), the 90/105 ompA gene (GENBANK®Accession no. AY762608), and the 7778B15 ompA gene (GENBANK® Accessionno. AY762612). C. psittaci primer sequences and expected amplicon sizesare listed in Table 1. Primer set Ppac was designed to amplify all C.psittaci genotypes, while primer set GTpc was designed to specificallyamplify only C. psittaci genotypes. The Ppac assay demonstrated 96%efficiency, while the GTpc assay displayed 99% efficiency, calculatedusing a standardized dilution series of quantitated DNA of C. psittacitested in triplicate over 6 logs (200 pg to 2 fg). The average for thisdata is reported as the square of the coefficient of regression values(efficiency); both assays (Ppac and GTpc) had a lower limit of detectionof at least 200 fg. A specific C. caviae marker targeting the ompA geneof C. caviae (GENBANK® Accession no. AF269282) was designed using theabove-described primer chemistry with unlabeled C.cav-F(5′-CCGTTGCAGACAGGAATAACA-3′, SEQ ID NO: 17) and FAM-labeled C.cav-R(5′-cacaaaGCTAAGAAAGCCGCGTTTG“t”G-3′, SEQ ID NO: 16) (the 5′ lowercaseletters are not part of the primer but correspond to a self-quenchingcomplementary tail; “t” represents the FAM binding location). Theexpected amplicon size using the C. caviae specific primers is 78 bp.

Real-Time PCR and HRM Analysis

For development of the methods disclosed herein, novel oligonucleotideprimers were designed based on previously published ompA gene sequencesand sequences available in GENBANK® using D-LUX™ design software(Invitrogen, CA). The primers for the LUX™ real-time PCR assay weredesigned to amplify the ompA gene of C. psittaci. The C. psittaciforward real-time PCR primers were 3′-labeled with 6-carboxy-fluorescein(FAM) and possessed a 5′ self quenching moiety (hairpin structure). C.caviae primers were 3′-labeled with 6-carboxy-fluorescein (FAM) andpossessed a 5′-self quenching moiety (hairpin structure). Thecorresponding reverse primers were designed and were used along with thelabeled forward primer to amplify the target nucleic acid. A full listof primer sequences are provided in Table 1.

TABLE 1 Real-time PCR primers^(a) Amplicon  Oligonucleotide Sequencesize Ppac forward, 5′-gaacccTATTGTTTGCCGCTACGGGT“t”C-3′ 109 Labeled(SEQ ID NO: 3) Ppac reverse, 5′-TCCTGAAGCACCTTCCCACA-3′ unlabeled(SEQ ID NO: 4) GTpc forward, 5′-gaactcaTGTGCAACTTTAGGAGCTGAG“t”TC-3′ 274Labeled (SEQ ID NO: 5) GTpc reverse, 5′-GCTCTTGACCAGTTTACGCCAATA-3′unlabeled (SEQ ID NO: 6) GT-F forward,5′-gacgcCATTCGTGAACCACTCAGCG“t”C-3′ 98 Labeled (SEQ ID NO: 7)GT-F reverse, 5′-CTCCTACAGGAAGCGCAGCA-3′ unlabeled (SEQ ID NO: 8)C. cav-reverse,  (5′-cacaaaGCTAAGAAAGCCGCGTTTG“t”G-3′ 78 labeled(SEQ ID NO: 16) C. cav-forward,  5′-CCGTTGCAGACAGGAATAACA-3′ (SEQ IDunlabeled NO: 17) ^(a)The 5′ lowercase letters are not part of theprimer itself but correspond to a self-quenching, complementary tail. b,“t” represents the FAM binding location.

Utilization of LUX™ chemistry primers and HRM analysis was performed asfollows. The reaction mixture for all primer sets was prepared using aSuperMix-UDG platinum quantitative PCR kit (Invitrogen, CA) containingthe following components per reaction mixture: 12.5 μl of 2× master mix,final concentrations of 100 nM each of the forward and reverse primers,0.15 μl of platinum Taq polymerase (5 U/μl), 5 ng of a template, andnuclease-free water (Promega) added to give a final volume of 25 μl.Real-time PCR was performed with a Corbett Rotor-Gene 6000™ (CorbettLife Sciences) under the following cycling conditions: 1 cycle at 95° C.for 2 min, followed by 45 cycles at 95° C. for 5 seconds and 62° C. for15 seconds, with data acquired at the 62° C. step in the green channel.Following amplification, high-resolution melt analysis was performedbetween 75° C. and 85° C. in 0.05° C. increments, with fluorescencenormalization regions between 75.5° C. and 76° C., before separation andat 84° C. to 84.5° C. after the separation. All isolates were tested intriplicate. All specimens (avian and mammalian) were screened for thepresence of C. psittaci, followed by genotyping, if applicable.

High-resolution melt (HRM) curves are derived by selecting twonormalization regions, one occurring prior to the melting of thedouble-stranded product and one following complete separation of the twostrands. Each region is generated by default by the software associatedwith the instrument performing the high-resolution melt analysis, butmay be manipulated manually to achieve optimum results. Thenormalization regions function to normalize the florescence of the meltcurves from the raw channel by averaging all starting and endingfluorescence values such that the end point value of each sample isidentical to the average. This allows for the melting profile of eachisolate to be analyzed relative to other isolates, which is particularlyuseful when compared unrelated samples.

Sequencing

Amplification of the ompA gene from isolate strains (DD34, CP3, CT1,NJ1, Vr-122 and VS-225) and specimens (3, 5, 25, 30, 31 and 83) wasperformed using previously published primers (Kaltenboeck et al., 1993,J. Bacteriol, 175:487-502 which is incorporated herein by reference tothe extent that it discloses the previously identified primers) andnewly developed primers ompA-F (5′-ACTATGTGGGAAGGTGCT-3′) (SEQ ID NO: 9)and ompA-R (5′-TAGACTTCATTTTGTTGATCTGA-3′) (SEQ ID NO: 10). The PCRmixture was prepared using a SuperMix-UDG platinum quantitative PCR kit(Invitrogen, CA) containing the following components per reactionmixture: 5 μl of 10×PCR master mix-MgCl₂, 1.5 μl 50 mM MgCl₂, finalconcentrations of 100 nM each of the forward and reverse primers, 0.5 μlof platinum Taq polymerase (5 U/μl), 1 μl of 10 μM PCR nucleotidemixture (Promega, CO.), 5 ng of a template, and nuclease-free water(Promega, CO) added to give a final volume of 50 μl.

A DNA engine dyad peltier thermocycler (Bio-Rad, CA.) was used foramplification under the following cycling conditions: 1 cycle at 95° C.for 2 min, followed by 50 cycles at 95° C. for 1 min, 59° C. for 1 min,and 72° C. for 2 min. Amplified samples were purified using a QIAquickgel extraction kit (Qiagen, CA) after separation on a 1% agarose gel.Sequencing was performed with an ABI 3130XL instrument (AppliedBiosystems, Inc.) under standard conditions for an 80-cm capillary.Consensus sequences were generated using DNAstar Lasergene SeqMan Prosoftware and aligned with published ompA gene sequences for eachgenotype by using Clustal W software. The GENBANK® Accession numbersused for alignment are as follows: AY762608, AY762609, AF269261,AY762610, AY762611, AY762612, and AY762613.

Real-Time PCR Analytical Sensitivity and Specificity Determinations

For lower limit of detection (LLD) assessments, serial dilution over6-logs (equivalent to 200 pg to 2 fg) of quantitated C. psittaci DNA wasprepared and aliquots tested using the above real-time PCR protocols.Both the Ppac assay and the GTpc assay had a lower limit of detection ofat least 200 fg.

EXAMPLE 2 Specificity of LUX™ Primers to Detect and Identify C. psittaciNucleic Acids

This example shows the ability of the newly-developed C.psittaci-specific primers to detect C. psittaci. The species-specificityof the primers is also confirmed. The use of these primers to identifyChlamydophila species as well as differentiate between C. psittacigenotypes as described in this example is shown in FIG. 6.

Three C. psittaci primer pairs described herein (SEQ ID NOs: 3 and 4,SEQ ID NOs: 5 and 6, and SEQ ID NOs: 7 and 8) successfully amplifiedsequences from Ppac, GTpc, and GT-F (respectively) from referencestrains of C. psittaci using real-time PCR and HRM analysis. Thespecificity of these primers was determined by a lack of PCRamplification product using 15 ng of DNA template from a variety ofbacterial and viral agents (Table 2). Human DNA was also unreactive withthe tested primer pairs. The newly developed C. psittaci-specificprimers also lacked reactivity with the four Chlamydophila agentstested.

The results of the HRM analysis using the Ppac primers yielded similarmelt curves for each C. psittaci genotype (FIG. 3A). The Ppac primeramplified C. caviae but this amplification product was easilydistinguishable from C. psittaci nucleic acids by HRM analysis. Inparticular, the distinction of C. caviae nucleic acids from C. psittacinucleic acids was determined by the observation of a dissociation curveoccurring prior to the dissociation curve of C. psittaci (FIG. 3A). Incontrast, the dissociation curve of C. abortus obtained using the Ppacprimers was indistinguishable from the corresponding dissociation curveof C. psittaci (FIG. 3A).

HRM analysis of the amplified product from each isolate in combinationwith the GTpc primer set produced a distinct melt curve profile for allC. psittaci genotypes except genotypes D and F, which were separatedusing the C. psittaci F genotype-specific primer, GT-F (FIG. 3C).Separation among the C. psittaci genotypes occurs in incremental shifts,with genotype E being farthest right (dissociating at the highesttemperature), with C. psittaci genotypes A, B, D/F, and C alldissociating at progressively lower temperatures (FIG. 3B).

Additionally, C. caviae can be distinguished from C. psittaci genotypesusing the GTpc primer set and HRM analysis. The C. caviae referencestrain was found to melt between C. psittaci genotypes D/F and B;further verification was also achieved by using the additionalspecies-specific C. caviae primer set under standard RT-PCT conditions(SEQ ID NO: 16 and SEQ ID NO: 17). Samples containing C. psittaci wereamplified under real-time PCR conditions and an amplification plotdisclosing the threshold value was determined (FIG. 5A). In thisexperiment, none of the samples containing C. psittaci were amplified toa sufficient extent for the sample to be positively identified as C.caviae.

C. abortus cannot be differentiated from C. psittaci using the Ppacprimer set. However, the GTpc primer set does not amplify C. abortus andtherefore provides a method to eliminate C. abortus as a suspectedpathogen in a Chlamydophila sample, where the sample is successfullyamplified using the GTpc primer set.

TABLE 2 Specificity Panel^(a) Agents screened for reactivity withprimers: SEQ ID NOs: 3-8 and SEQ ID NO: 16 and 17: Candida albicansBordetella pertussis Chlamydophila felis Chlamydophila pecorumChlamydophila pneumoniae Chlamydia trachomatis Corynebacteriumdiphtheriae Coxiella burnetii Escherichia coli Haemophilus influenzaeLactobacillus planitarium Legionella longbeachae Legionella pneumophilaMoraxella catarrhalis Mycoplasma arginini Mycoplasma buccale Mycoplasmafaucium Mycoplasma fermentans Mycoplasma genitalium Mycoplasma hominisMycoplasma hyorhinis Mycoplasma lipophilum Mycoplasma orale Mycoplasmapenetrans Mycoplasma pirum Mycoplasma salivarium Mycobacteriumtuberculosis Neisseria elongata Neisseria meningitidis Pseudomonasaeruginosa Staphylococcus aureus Staphylococcus epidermidisStreptococcus pneumoniae Streptococcus pyogenes Streptococcus salivariusUreaplasma urealyticum Human DNA Adenovirus Coronavirus Parainfluenzavirus 2 Parainfluenza virus 3 Rhinovirus Influenza virus A Influenzavirus B Respiratory syncytial virus A Respiratory syncytial virus B^(a)Shown are bacterial and viral species (15 ng) screened forcross-reactivity by using the disclosed real-time PCR assay.All agents listed were undetected (no amplification).

EXAMPLE 3 Specimen Testing

This example shows the use of the disclosed primers to screen DNAspecimens for the presence of C. psittaci and C. caviae. The genotypesof the C. psittaci identified in the specimens were also determined.

One hundred sixty-nine specimens obtained from birds and companionmammals were screened along with reference strains. Of these archivednucleic acid preparations, 107 (63.3%) were positive for chlamydial DNA,98 (91.6%) were positive for C. psittaci, and 9 (8.4%) were positive forC. caviae. Of the positive C. psittaci samples, 70 (71.4%) were genotypeA, 3 (3.1%) were genotype B, 4 (4.1%) were genotype E, and 21 (21.4%)were positive for C. psittaci (positive amplification for both Ppac andGTpc markers) but could not be typed using this assay, due toinconclusive melt curve data (Table 3). All C. caviae strains wereobtained from guinea pig specimens.

TABLE 3 Real-time PCR and HRM genotyping results for avian and mammalianspecimens^(a) Specimen No. Specimen Origin Bacterial Sp.^(b) Genotype 1Lovebird * A 2 Cockatiel * A 3 Macaw * E 4 Lovebird * A 5 Cockatiel * E6 Sun conure * A 7 Sun conure * A 8 Sun conure * A 9 Sun conure * A 10Sun conure * A 11 Sun conure * A 12 Sun conure * A 13 Cockatiel * A 14Cockatiel * A 15 Parakeet * A 16 Cockatiel * A 17 Not given * A 18Amazon * A 19 Macaw * A 20 Parakeet * A 21 Green cheek * A 22 Greencheek * A 23 Cockatiel * A 24 Pooled cockatiels * A 25 Cockatiel * A 26Cockatiel * A 27 Amazon * A 28 Cockatiel * A 29 Lovebird * A 30 Pigeon *B 31 Pigeon * B 32 Cockatiel * A 33 Cockatiel * A 34 Cockatiel * A 35Cockatiel * A 36 Guinea pig C. caviae 37 Yellow-naped Amazon * A 38Pionus * A 39 Cockatiel * A 40 Guinea pig C. caviae 41 Cockatiel * A 42Cockatiel * A 43 Cockatiel * A 44 Guinea pig C. caviae 45 Guinea pig C.caviae 46 Lori * B 47 Lovebird * A 48 Amazon * A 49 Hawkhead * A 50Ringneck * A 51 Conure * A 52 Macaw * A 53 African gray * A 54Cockatiel * A 55 Cockatiel * A 56 Cockatiel * A 57 Guinea pig C. caviae58 Guinea pig C. caviae 59 Sun conure * A 60 Guinea pig C. caviae 61Pigeon * E 62 Cockatiel * A 63 Guinea pig C. caviae 64 Cockatiel * A 65Amazon * A 66 DYH Amazon * A 67 Cockatiel * A 68 Guinea pig C. caviae 69Cockatiel * A 70 Cockatiel * A 71 Amazon * A 72 Amazon * A 73 Amazon * A74 Cockatiel * 75 Cockatiel * A 76 Brown crown * A 77 Cockatiel * A 78Lovebird * A 79 Cockatiel * A 80 Cockatiel * A 81 Cockatiel * E 82Macaw * A 83 Parrotlet * A 84 Cockatiel * A 85 Amazon * A 86 Amazon * A^(a)Results of real-time PCR testing and HRM analysis are shown for 86chlamydia-positive avian and mammalian specimens. ^(b)* indicates C.psittaci

EXAMPLE 4 Sequencing Analysis

The ompA gene was sequenced for all available reference strains and asubset of C. psittaci-positive specimens that contained nucleic acids insufficient amounts and of sufficient quality, acquired from theInfectious Diseases Laboratory, University of Georgia, as describedabove. The data shows that methods of identifying C. psittaci describedherein correctly identified DD34 and specimens 25 and 83 as genotype A,CP3 and specimens 30 and 31 as genotype B, CT1 as genotype C, NJ1 asgenotype D, Vr-122 and specimens 3 and 5 as genotype E, and VS-225 asgenotype F. The sequence alignment of the target regions for this assaywithin the ompA gene of C. psittaci genotypes A-F are shown in FIGS. 4Aand 4B. Alignment of the Ppac amplicon sequences yielded the consensussequence set forth as SEQ ID NO: 18. Of C. psittaci genotypes A-F, onlygenotype C had had a variation within the Ppac amplicon, which is setforth as SEQ ID NO: 19. Alignment of the GTpc amplicons of C. psittacigenotypes A-F also yielded a consensus sequence (SEQ ID NO: 20).However, considerably more divergence was observed between the sixgenotypes. (SEQ ID NOs: 21-26). The nucleotide sequences of the Ppacamplicon consensus, Ppac amplicons from genotype C, GTpc ampliconconsensus (with non-consensus positions indicated by “N”), and GTpcamplicons from genotypes A-F are as follows:

Ppac amplicon consensus (SEQ ID NO: 18)ATCGGCATTATTGTTTGCCGCTACGGGTTCCGCTCTCTCCTTACAAGCCTTGCCTGTAGGGAACCCAGCTGAACCAAGTTTATTAATCGATGGCACTA TGTGGGAAGGTGCTTCAGGAGAPpac C. psittaci genotype C amplicon (SEQ ID NO: 19)ATCGGCATTATTATTTGCCGCTACGGGTTCCGCTCTCTCCTTACAAGCCTTGCCTGTAGGGAACCCAGCTGAACCAAGTTTATTAATCGATGGCACTA TGTGGGAAGGTGCTTCAGGAGAGTpc amplicon consensus (SEQ ID NO: 20)GGGAATGTGGTTGTGCAACTTTAGGAGCTGAGTTCCAATACGCTCAATCTAATCCTAANATTGAAATGCTCAANGTNACTTCAAGCCCAGCACAATTTGTGATTCACAAACCAAGAGGCTATAAAGGANCTNGCTCGAATTTTCCTTTACCTATAACNGCTGGNACANNNGNNGCTACAGANACNAAATCNGCNACANTNAAATANCATGAATGGCAAGTNGGNCTNGCNCTNTCTTACAGATTGAANATGCTTGTTCCNTANATTGGCGTAAACTGGTCAAGAGCAACTTTTG ATGCTGGTpc C. psittaci genotype A amplicon (SEQ ID NO: 21)GGGAATGTGGTTGTGCAACTTTAGGAGCTGAGTTCCAATACGCTCAATCTAATCCTAAGATTGAAATGCTCAACGTCACTTCAAGCCCAGCACAATTTGTGATTCACAAACCAAGAGGCTATAAAGGAGCTAGCTCGAATTTTCCTTTACCTATAACGGCTGGAACAACAGAAGCTACAGACACCAAATCAGCTACAATTAAATACCATGAATGGCAAGTAGGCCTCGCCCTGTCTTACAGATTGAATATGCTTGTTCCATATATTGGCGTAAACTGGTCAAGAGCAACTTTTG ATGCTGGTpc C. psittaci genotype B amplicon (SEQ ID NO: 22)GGGAATGTGGTTGTGCAACTTTAGGAGCTGAGTTCCAATACGCTCAATCTAATCCTAAGATTGAAATACTCAACGTCACTTCAAGCCCAGCACAATTTGTGATTCACAAACCAAGAGGCTATAAAGGAGCTAGCTCGAATTTTCCTTTACCTATAACGGCTGGAACAACAGAAGCTACAGACACCAAATCAGCTACAATTAAATACCATGAATGGCAAGTAGGCCTCGCCCTGTCTTACAGATTGAATATGCTTGTTCCATATATTGGCGTAAACTGGTCAAGAGCAACTTTTG ATGCTGGTpc C. psittaci genotype C amplicon (SEQ ID NO: 23)GGGAATGTGGTTGCGCAACTTTAGGAGCTGAATTCCAATACGCTCAATCTAATCCTAAAATTGAAATGTTGAATGTAATCTCCAGCCCAGCACAATTTGTGGTTCACAAGCCTAGAGGATACAAGGGAACGTCCGCCAACTTTCCTTTACCTGCAAATGCAGGCACAGAGGCTGCTACGGATACTAAATCTGCAACACTCAAATATCATGAATGGCAAGTTGGTCTAGCACTCTCTTACAGATTGAACATGTTAGTTCCTTACATTGGCGTAAACTGGTCACGAGCAACTTTTG ATGCCGGTpc C. psittaci genotype D amplicon (SEQ ID NO: 24)GGGAATGTGGTTGTGCGACTTTAGGAGCCGAGTTCCAATACGCTCAATCTAATCCTAAAATTGAAATGCTCAATGTAACTTCAAGCCCAGCACAATTTGTGATTCACAAACCAAGAGGCTATAAAGGAACTGGCTCGAATTTTCCTTTACCTATAGACGCGGGTACAGAGGCTGCTACAGATACTAAGTCTGCAACACTCAAATATCATGAATGGCAAGTTGGTCTAGCACTCTCTTACAGATTGAACATGCTTGTTCCTTACATTGGCGTAAACTGGTCAAGAGCAACTTTTG ATGCTGGTpc C. psittaci genotype E amplicon (SEQ ID NO: 25)GGGAATGTGGTTGTGCAACTTTAGGAGCTGAGTTCCAATACGCTCAATCTAATCCTAAGATTGAAGTGCTCAACGTCACTTCAAGCCCAGCACAATTTGTGATTCACAAACCAAGAGGCTATAAAGGAGCTAGCTCGAATTTTCCTTTACCTATAACGGCTGGAACAACAGAAGCTACAGACACCAAATCAGCTACAATTAAATACCATGAATGGCAAGTAGGCCTCGCCCTGTCTTACAGATTGAATATGCTTGTTCCATATATTGGCGTAAACTGGTCAAGAGCAACTTTTG ATGCTGGTpc C. psittaci genotype F amplicon (SEQ ID NO: 26)GGGAATGTGGTTGTGCAACTTTAGGAGCTGAATTCCAGTATGCTCAATCTAATCCTAAAATTGAAATGCTGAATGTAATCTCCAGCCCAACACAATTTGTAGTTCACAAGCCTAGAGGATACAAGGGAACAGGATCGAACTTTCCTTTACCTCTAACAGCTGGTACAGATGGTGCTACAGATACTAAATCTGCAACACTCAAATATCATGAATGGCAAGTTGGTTTAGCGCTCTCTTACAGATTGAACATGCTTGTTCCTTACATTGGCGTAAACTGGTCAAGAGCAACTTTTG ATGCTG

EXAMPLE 5 Detection of C. psittaci, C. abortus, and C. caviae andGenotyping of C. psittaci

This example shows the development and implementation of a noveldiagnostic method that is capable of rapidly detecting C. psittaci, C.abortus, and C. caviae in a sample and also rapidly genotyping C.psittaci. The methods used in this example are shown schematically inFIG. 6A (identification of C. psittaci, C. abortus, and C. caviae) andFIG. 6B (genotyping C. psittaci).

Three novel primer sets (Ppac (SEQ NOs: 3 and 4), GTpc (SEQ NOs: 5 and6), and GT-F (SEQ NOs: 7 and 8)), with the use of standard cyclingconditions followed by HRM, are able to reliably identify anddifferentiate C. psittaci genotypes A through F and detect the closelyrelated C. abortus and C. caviae species. The Ppac primers (SEQ NOs: 3and 4) serve as pan markers, able to amplify all C. psittaci genotypesas well as C. caviae and C. abortus, while the GTpc primers (SEQ NOs: 5and 6) are capable of distinguishing C. caviae and all C. psittacigenotypes except D/F. The GT-F (SEQ NOs: 7 and 8) primers can be used tospecifically amplify genotype F, thus providing a comprehensivealgorithm for identifying and genotyping C. psittaci in instances whereHRM analysis detects a characteristic genotype D/F melt curve. All threeprimer sets also demonstrate specificity for their respective nucleictargets and sensitivity to at least 200 fg with C. psittaci referencestrain DNA.

The method was successfully used on clinical specimens and displayed100% concordance with sequence data generated from both referenceisolates and clinical extracts. Numerous avian specimens screened werepositive for C. psittaci. Genotype A was the most frequently identifiedgenotype, present in 71.4% of the C. psittaci-positive specimens thatcould be typed. Reportedly, genotype A is commonly identified inchlamydia-positive psittacine birds, such as parrots, cockatoos, andcockatiels and the instant findings are therefore consistent withprevious reports. Both the B and the E genotypes of C. psittaci wererarely present in the clinical samples, accounting for only 7.2% of thespecimens. Historically, these genotypes have most commonly beenidentified in pigeons and doves, again consistent with the results ofthe instant assay. Notably, C. psittaci genotypes C, D, and F were notfound in any of the specimens tested, supporting the claim by othersthat the vast majority of naturally occurring genotypes belong to theA/B/E cluster. C. caviae was detected only in guinea pig specimens(Table 3), where it is known to exist. No specimens tested positive forC. abortus, an agent typically found in mammals, although a fewpsittacine infections have been reported. Neither C. caviae nor C.abortus is considered a classic respiratory pathogen and would notlikely be present in respiratory samples being tested for C. psittaci inhumans. However, if amplification with Ppac primers (SEQ NOs: 3 and 4)yielded a positive amplification curve while amplification of the samesample or source using the GTpc primers (SEQ NOs: 5 and 6) provided anegative result, C. abortus should be considered as the potentialpathogen and ompA sequencing should be performed for verification.Similarly, amplification with both Ppac primers (SEQ NOs: 3 and 4) andthe C. caviae specific primer pair (SEQ ID NOs: 16 and 17) indicates thepresence of C. caviae.

The reliability of HRM analysis is dependent upon both sufficientquantity and sufficient quality of the starting template. As such,specimens with amplification curves that are not sigmoidal or havethreshold cycle values of 40 or above should use melt curve data. Ifpossible, these samples should be concentrated and retested forverification. When threshold cycle values of less than 40 are achieved,the HRM data was found to be remarkably consistent, reproducible, andreliable, as evidenced by the substantially identical melt curvesgenerated in each of the triplicate samples assayed on numerousoccasions and verified by subsequent sequence analysis. The quality andamount of template nucleic acid are inherent limitations in anyreal-time PCR assay but are particularly important when HRM analysis isperformed. These limitations may account for the 21% of the specimensthat were unable to be definitively genotyped using the disclosedmethods since some older specimens may not have been properly storedafter submission. FIG. 5B is an amplification plot of samples providedby the University of Georgia that tested C. psittaci positive on initialexamination. In this experiment, the archived samples were subjected tostandard real-time PCR conditions and a threshold value determined. Asseen in FIG. 5B, the majority of the nucleic acid samples were positivefor the amplification of C. psittaci nucleic acids. The samples belowthe threshold are C. psittaci negative.

EXAMPLE 6 Amplification of Chlamydophila Nucleic Acids with a Specificand a Universal Primer Sequence

The above examples describe indirect detection of a Chlamydophilaspecies by amplification of Chlamydophila nucleic acids using thedefined primer disclosed herein. This example describes amplification ofChlamydophila nucleic acids using a one defined primer and one universalprimer.

To amplify C. psittaci nucleic acids with one defined and one universalprimer, the nucleic acids in the sample from a subject may be pretreatedto add a repeat sequence to the end of the DNA in the sample, forexample, using terminal transferase to add a repeat sequence to the 3′end of the DNA in the sample. Then one forward primer comprising forexample SEQ ID NO: 3 is used in conjunction with a universal primer thatis complementary to the repeat sequence added by terminal transferase tpamplify the nucleic acids in the sample. The detection of amplifiednucleic acids indicates the presence of C. psittaci in the sample.

EXAMPLE 7 Direct Detection of a Chlamydophila Infection

Methods of indirect detection of Chlamydophila species in a sample byPCR amplification of C. psittaci, C. abortus, and C. caviae nucleicacids in a sample are described above. This example illustrates directdetection of C. psittaci, C. abortus, and C. caviae by hybridization ofa labeled primer to a target nucleic acid in a sample.

To directly detect a C. psittaci infection in a subject, a nucleic acidsample from a subject containing or suspected of containing C. psittaciis contacted with at least one labeled primer comprising the sequence ofany one of SEQ ID NOs: 3-8 under very high stringency hybridizationconditions. The sample may be contacted by the labeled probe in anyhybridization assay known to the art, such as a dot blot assay.Detection of hybridization of the probe to target nucleic acids in thesample will indicate the presence of C. psittaci nucleic acids in thesample and positively diagnose an C. psittaci infection.

One of skill will recognize that this technique can also be used toidentify a C. caviae infection (detection of hybridization of probescomprising SEQ ID NOs: 16 or 17), a C. psittaci genotype F infection(detection of hybridization of probes comprising SEQ ID NOs: 7 or 8), ora C. abortus infection (detection of hybridization of probes comprisingSEQ ID NOs: 3 or 4 but not SEQ ID NOs: 5 or 6).

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

The invention claimed is:
 1. A method for diagnosing a Chlamydophilapsittaci, Chlamvdophila caviae, or Chlamvdophila abortus infection in asubject suspected of having a Chlamydophila psittaci, Chlamvdophilacaviae, or Chlamvdophila abortus infection, comprising: obtaining fromthe subject a sample comprising nucleic acids; contacting the samplewith a first primer pair comprising a forward primer consisting of thenucleic acid sequence set forth as SEQ ID NO: 3 and a label and areverse primer consisting of the nucleic acid sequence set forth as SEQID NO: 4 and a second primer pair comprising a forward primer consistingof the nucleic acid sequence set forth as SEQ ID NO: 5 and a label and areverse primer consisting of the nucleic acid sequence set forth as SEQID NO: 6; amplifying nucleic acids in the sample using said first andsecond primer pair to form amplified Chlamydophila nucleic acids; anddetecting the amplified Chlamydophila nucleic acids and identifying thepresence or absence of amplified Chlamydophila psittaci, Chlamydophilacaviae, or Chlamydophila abortus nucleic acids in the amplifiedChlamydophila nucleic acids, thereby diagnosing a Chlamydophilapsittaci, Chlamydophila caviae, or Chlamydophila abortus infection inthe subject.
 2. The method of claim 1, further comprising amplifyingnucleic acids in the sample using a third primer pair comprising aforward primer consisting of the nucleic acid sequence set forth as SEQID NO: 7 and a label and a reverse primer consisting of the nucleic acidsequence set forth as SEQ ID NO: 8; and detecting the presence orabsence of amplified nucleic acids, wherein the presence of amplifiednucleic acids indicates that the subject is infected with Chlamydophilapsittaci genotype F, and wherein the absence of amplified nucleic acidsindicates that the subject is infected with Chlamydophila psittacigenotype D.
 3. The method of claim 1, further comprising discriminatingbetween a Chlamydophila psittaci infection and a Chlamydophila caviaeinfection by a process comprising: contacting the sample with aChlamydophila caviae-specific primer that hybridizes under very highstringency conditions to the complement of a Chlamydophila caviaenucleic acid sequence set forth as SEQ ID NO: 16 and a Clamydophilacaviae-specific primer that hybridizes under very high stringencyconditions to the complement of a Chlamydophila caviae nucleic acidsequence set forth as SEQ ID NO: 17, amplifying Chlamydophila caviaenucleic acids in the sample to form amplified Chlamydophila caviaenucleic acids; and detecting the amplified Chlamydophila caviae nucleicacids, wherein detection of the amplified Chlamydophila caviae nucleicacids indicates that the subject is infected with Chlamydophila caviae.4. The method of claim 1, further comprising contacting the sample witha primer pair comprising a forward primer consisting of the nucleic acidsequence set forth as SEQ ID NO: 16 and a label and a reverse primerconsisting of the nucleic acid sequence set forth as SEQ ID NO: 17;amplifying Chlamydophila caviae nucleic acids in the sample to formamplified Chlamydophila caviae nucleic acids; and detecting theamplified Chlamydophila caviae nucleic acids, wherein detection of theamplified Chlamydophila caviae nucleic acids indicates that the subjectis infected with Chlamydophila caviae.
 5. The method of claim 1, furthercomprising discriminating between a Chlamydophila psittaci infection anda Chlamydophila caviae infection by a process comprising: performinghigh resolution melt analysis of the amplified nucleic acids; anddetecting the presence of a Chlamydophila caviae-specific amplificationproduct with the high resolution melt analysis, wherein the presence ofthe Chlamydophila caviae-specific amplification product is indicative ofa Chlamydophila caviae infection.
 6. The method of claim 1, furthercomprising distinguishing between Chlamydophila psittaci genotypes A, B,C, D/F and E, by a process comprising: performing high resolution meltanalysis of the amplified nucleic acids; and detecting the presence of aChlamydophila psittaci genotype A, B, C, or E-specific amplificationproduct with the high resolution melt analysis, wherein the presence ofthe Chlamydophila psittaci genotype A, B, C, or E-specific amplificationproduct indicates that the subject is infected with Chlamydophilapsittaci genotype A, B, C, or E, respectively.
 7. The method of claim 6,further comprising distinguishing between Chlamydophila psittacigenotypes D and F by a process comprising: contacting the sample with athird primer pair comprising a forward primer consisting of the nucleicacid sequence set forth as SEQ ID NO: 7 and a label and a reverse primerconsisting of the nucleic acid sequence set forth as SEQ ID NO: 8;amplifying Chlamydophila psittaci genotype F nucleic acid in the sampleto form amplified Chlamydophila psittaci genotype F nucleic acids; anddetecting the amplified Chlamydophila psittaci genotype F nucleic acids,wherein detection of the amplified Chlamydophila psittaci genotype Fnucleic acids indicates that the subject is infected with Chlamydophilapsittaci genotype F, and wherein a lack of amplified Chlamydophilapsittaci genotype F nucleic acids indicates that subject is infectedwith Chlamydophila psittaci genotype D.
 8. The method of claim 1,wherein the subject is an avian subject or a mammalian subject.
 9. Themethod of claim 8, wherein the mammalian subject is a human subject.